IMPROVED LENTIVIRAL EXPRESSION VECTOR, CONSTRUCTION METHOD FOR SAME, AND APPLICATIONS THEREOF

Abstract

Provided are an improved lentiviral expression vector, a construction method for same, and applications thereof. The plasmid backbone of the lentiviral expression vector is based on pLVX-Puro and is improved via the following steps: a. adding a CMV enhancer and promoter to the upstream of 5′ LTR by means of gene synthesis and subcloning; b. replacing the original 5′ LTR with a new 5′ LTR (SEQ ID NO: 3) of which the length is 181 bps; c. replacing the CMV promoter on an original vector with an EFS promoter; d. deleting a PGK promoter after multiple cloning sites of the original vector and, at the same time, introducing a P2A connection subsequence; e. replacing original resistance against Puro with resistance against a shorter Blasticidin; and f. replacing original 3′ LTR with an LTR (SEQ ID NO: 7) with a partially deleted U3. The lentiviral expression vector acquired by the improvement provides a greater packaging capacity in comparison with pLVX-Puro.

Claims

1: A lentiviral expression vector improved on the basis of PLVX-Puro, wherein the vector comprises at least one, at least two or three of the following improved element sequences: (1) a CMV promoter sequence upstream of 5′ LTR, (2) an EFS promoter sequence as a target gene promoter, and (3) a P2A linker sequence downstream of a multiple cloning site.

2: The lentiviral expression vector according to claim 1, further comprising a CMV enhancer sequence upstream of the CMV promoter sequence upstream of the 5′ LTR.

3: The lentiviral expression vector according to claim 1, wherein the 5′ LTR is a truncated PLVX-Puro 5′ LTR sequence.

4: The lentiviral expression vector according to claim 1, further comprising a 3′ LTR sequence with a partially deleted U3 region.

5: The lentiviral expression vector according to claim 1, wherein the CMV promoter sequence upstream of the 5′ LTR comprises a nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence that has at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 2 and has a promoter activity.

6: The lentiviral expression vector according to claim 1, wherein the EFS promoter sequence comprises a nucleotide sequence shown in SEQ ID NO: 4 or a nucleotide sequence that has at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 4 and has a promoter activity.

7: The lentiviral expression vector according to claim 1, wherein the P2A linker sequence comprises a nucleotide sequence shown in SEQ ID NO: 5 or a nucleotide sequence that has at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 5 and has a ligation function.

8: The lentiviral expression vector according to claim 2, wherein the CMV enhancer sequence comprises a nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence that has at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 1 and has an enhancer activity.

9: The lentiviral expression vector according to claim 3, wherein the truncated 5′ LTR sequence comprises a nucleotide sequence shown in SEQ ID NO: 3 or a nucleotide sequence that has at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 3 and can function as the 5′ LTR.

10: The lentiviral expression vector according to claim 4, wherein the 3′ LTR sequence with the partially deleted U3 region comprises a nucleotide sequence shown in SEQ ID NO: 7 or a nucleotide sequence that has at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 7 and can function as the 3′ LTR.

11: The lentiviral expression vector according to claim 1, wherein the vector further comprises a Blasticidin resistance gene sequence in place of a Puro resistance gene sequence.

12: The lentiviral expression vector according to claim 11, wherein the Blasticidin resistance gene sequence comprises a nucleotide sequence shown in SEQ ID NO: 6 or a nucleotide sequence that has at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 6 and has resistance.

13: A method of improving a lentiviral expression vector on the basis of PLVX-Puro, the method comprising: (1) adding a CMV promoter sequence to the upstream of 5′ LTR, (2) replacing a CMV promoter of a target gene with an EFS promoter sequence, and (3) deleting a PGK promoter downstream of a multiple cloning site and introducing a P2A linker sequence.

14: The method according to claim 13, further comprising one or more of the following improving steps: (1) adding a CMV enhancer sequence to the upstream of the 5′ LTR, (2) replacing the original 5′ LTR with a truncated 5′ LTR sequence, and (3) replacing original 3′ LTR with a 3′ LTR sequence with a partially deleted U3 region.

15: The method according to claim 13, further comprising replacing a Puro resistance gene with a Blasticidin resistance gene sequence.

16: The method according to claim 13, wherein the CMV promoter sequence added comprises a nucleotide sequence that has at least 80% identity to a nucleotide sequence shown in SEQ ID NO: 2 and has a promoter activity; the EFS promoter sequence comprises a nucleotide sequence that has at least 80% identity to a nucleotide sequence shown in SEQ ID NO: 4 and has a promoter activity; and/or the P2A linker sequence comprises a nucleotide sequence that has at least 80% identity to a nucleotide sequence shown in SEQ ID NO: 5 and has a ligation function.

17: The method according to claim 14, wherein the CMV enhancer sequence added comprises a nucleotide sequence that has at least 80% identity to a nucleotide sequence shown in SEQ ID NO: 1 and has an enhancer function; the truncated 5′ LTR sequence comprises a nucleotide sequence that has at least 80% identity to a nucleotide sequence shown in SEQ ID NO: 3 and can function as the 5′ LTR; and/or the 3′ LTR sequence with the partially deleted U3 region comprises a nucleotide sequence that has at least 80% identity to a nucleotide sequence shown in SEQ ID NO: 7 and can function as the 3′ LTR.

18. (canceled)

19: A eukaryotic cell transfected with the lentiviral expression vector according to claim 1.

20. (canceled)

21: Use of the lentiviral expression vector according to claim 1, in the preparation of lentiviral particles.

22. (canceled)

23: Use of the lentiviral expression vector according to claim 1, for preparing a gene or cell therapy drug.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIGS. 1A and 1B are an original Plvx-Puro lentiviral expression vector and an improved lentiviral expression vector, respectively.

[0049] FIGS. 2A to 2E show maps of expression vectors into which target genes EGFR, Cas9, Cas9+EGFP, CCR2+Cas9+EGFP and EGFR+Cas9 are inserted, respectively.

[0050] FIGS. 3A to 3C are respectively experimental result diagrams of fluorescence expression of vectors into which target genes (EGFR, Cas9+EGFP, and CCR2+EGFP+Cas9) in different lengths are inserted, in cells, as detected by FACS.

DETAILED DESCRIPTION OF EMBODIMENTS

[0051] The embodiments of the present invention are described below by particular specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the disclosure of the specification. The present invention can also be implemented or applied by other different specific embodiments, and various details in the specification can also be modified or altered on the basis of different viewpoints and applications without departing from the spirit of the present invention.

[0052] Before the specific embodiments of the present invention are further described, it should be understood that the scope of protection of the present invention is not limited to the following particular specific embodiments. It should also be understood that the terms used in the examples of the present invention are intended to describe the particular specific embodiments, rather than limiting the scope of protection of the present invention, and have meanings commonly understood by those skilled in the art. In the specification and claims of the present invention, unless otherwise clearly stated in the context, the singular forms “a”, “an”, and “the” include the plural forms.

[0053] When numerical ranges are given in the examples, it should be understood that unless otherwise indicated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the examples, according to the knowledge in the prior art and the disclosure of the present invention, those skilled in the art can also use any prior art methods, devices, and materials which are similar or equivalent to the methods, devices, and materials described in the examples of the present invention to realize the present invention.

Example 1. Construction of Improved Lentiviral Expression Vector Backbone

[0054] The plasmid backbone of the present invention is based on PLVX-Puro (Takara, 632164, see the map of FIG. 1A), and is improved via the following steps:

a. adding a CMV enhancer (SEQ ID NO: 1) and promoter (SEQ ID NO: 2) to the upstream of 5′ LTR by means of gene synthesis and subcloning;
b. replacing the original 5′ LTR with a shorter new 5′ LTR (SEQ ID NO: 3) of which the length is 181 bps;
c. replacing the CMV promoter on an original vector with a broader-spectrum and shorter EFS promoter (SEQ ID NO: 4) of which the length is 256 bps:
d. deleting a PGK promoter downstream of a multiple cloning site of the original vector and, at the same time, introducing a P2A linker sequence (SEQ ID NO: 5);
e. replacing original resistance gene against Puro with a shorter resistance gene against Blasticidin (SEQ ID NO: 6) of which the length is 399 bps; and
f. replacing original 3′ LTR with an LTR (SEQ ID NO: 7) with a partially deleted U3 in order to obtain higher safety.

[0055] The specific experimental steps were as follows:

Vector-Improving Steps:

[0056] 1.1 A CMV enhancer, a CMV promoter and a new 5′LTR sequence were amplified to obtain a PCR product. The PCR product was subjected to electrophoresis detection, a band having a size consistent with the expected size of the target sequence was cut off, and the product was recovered for ligation and transformation. The amplified PCR product above was ligated with PLVX-puro linearized with PvuI/MfeI (PvuI: NEB, R3150GS; MfeI: NEB, R3589S) by using GenBuilder (GenScript, M00712) to obtain a ligated product. The ligated product was transformed into TOP10 (GenScript, NA) competent cells by a thermal shock method, the transformed product was evenly spread on an LB plate culture medium (GenScrinpt, NA) containing ampicillin resistance with a spreader, and the plate was inversely placed in a 37° C. incubator for overnight culture. A single colony was picked with a sterile pipette tip as a PCR template for colony screening, and transferred to an LB liquid culture medium (GenScript, NA) containing ampicillin resistance for culture. The PCR product for bacterial detection was subjected to electrophoresis detection, and a clone having a band with the size consistent with the expected size was selected for bacterial shaking and plasmid extraction.

[0057] 1.2 An EFS promoter sequence was amplified to obtain a PCR product. The PCR product was subjected to electrophoresis detection, a band having a size consistent with the expected size of the target sequence was cut off, and the product was recovered for ligation and transformation. The amplified PCR product above was ligated with the plasmid obtained in step 1.1 which is linearized with ClaI/XhoI (ClaI: NEB, R0197V; XhoI: NEB, R0146L) by using GenBuilder (GenScript, M00712) to obtain a ligated product. The ligated product was transformed into TOP10 (GenScript, NA) competent cells by a thermal shock method, the transformed product was evenly spread on an LB plate culture medium (GenScript, NA) containing ampicillin resistance with a spreader, and the plate was inversely placed in a 37° C. incubator for overnight culture. A single colony was picked with a sterile pipette tip as a PCR template for colony screening, and transferred to an LB liquid culture medium (GenScript, NA) containing ampicillin resistance for culture. The PCR product for bacterial detection was subjected to electrophoresis detection, and a clone having a band with the size consistent with the expected size was selected for bacterial shaking and plasmid extraction.

[0058] 1.3 A P2A sequence was amplified to obtain a PCR product. The PCR product was subjected to electrophoresis detection, a band having a size consistent with the expected size of the target sequence was cut off, and the product was recovered for ligation and transformation. The amplified PCR product above was ligated with the plasmid obtained in step 1.2 which is linearized with XbaI/BSiW (XbaI: NEB, R0145S; BSiW: NEB, R3553S) by using GenBuilder (GenScript, M00712) to obtain a ligated product. The ligated product was transformed into TOP10 (GenScript, NA) competent cells by a thermal shock method, the transformed product was evenly spread on an LB plate culture medium (GenScript, NA) containing ampicillin resistance with a spreader, and the plate was inversely placed in a 3T′C incubator for overnight culture. A single colony was picked with a sterile pipette tip as a PCR template for colony screening, and transferred to an LB liquid culture medium (GenScript, NA) containing ampicillin resistance for culture. The PCR product for bacterial detection was subjected to electrophoresis detection, and a clone having a band with the size consistent with the expected size was selected for bacterial shaking and plasmid extraction.

[0059] 1.4 A Blasticidin sequence was amplified to obtain a PCR product. The PCR product was subjected to electrophoresis detection, a band having a size consistent with the expected size of the target sequence was cut off, and the product was recovered for ligation and transformation. The amplified PCR product above was ligated with the plasmid obtained in step 1.3 which is linearized with BSU36I/XbaI (BSU36I: NEB, R0524V; XbaI: NEB, R0145S) by using GenBuilder (GenScript, M00712) to obtain a ligated product. The ligated product was transformed into TOP10 (GenScript, NA) competent cells by a thermal shock method, the transformed product was evenly spread on an LB plate culture medium (GenScript. NA) containing ampicillin resistance with a spreader, and the plate was inversely placed in a 37° C. incubator for overnight culture. A single colony was picked with a sterile pipette tip as a PCR template for colony screening, and transferred to an LB liquid culture medium (GenScript, NA) containing ampicillin resistance for culture. The PCR product for bacterial detection was subjected to electrophoresis detection, and a clone having a band with the size consistent with the expected size was selected for bacterial shaking and plasmid extraction.

[0060] 1.5 A 3′ LTR sequence was amplified to obtain a PCR product. The PCR product was subjected to electrophoresis detection, a band having a size consistent with the expected size of the target sequence was cut off, and the product was recovered for ligation and transformation. The amplified PCR product above was ligated with the plasmid obtained in step 1.4 which is linearized with BSU36I/StuI (BSU361: NEB, R0524V; StuI: NEB, R0187S) by using GenBuilder (GenScript, M00712) to obtain a ligated product. The ligated product was transformed into TOP10 (GenScript, NA) competent cells by a thermal shock method, the transformed product was evenly spread on an LB plate culture medium (GenScript. NA) containing ampicillin resistance with a spreader, and the plate was inversely placed in a 37° C. incubator for overnight culture. A single colony was picked with a sterile pipette tip as a PCR template for colony screening, and transferred to an LB liquid culture medium (GenScript. NA) containing ampicillin resistance for culture. The PCR product for bacterial detection was subjected to electrophoresis detection, and a clone having a band with the size consistent with the expected size was selected for bacterial shaking and plasmid extraction.

[0061] By means of the improvement for PLVX-Puro above, an improved lentiviral expression vector (Genscript-Lenti vector) is obtained, and the map is as shown in FIG. 1B.

Example 2. Verification of Gene Expression Function of Improved Lentiviral Expression Vector

2.1 Construction of Improved Lentiviral Expression Vector

[0062] In the experiment, five genes with different lengths as shown in Table 1 were selected, and each gene was subcloned into the improved lentiviral expression vector backbone in Example 1.

[0063] The steps of subcloning different target genes were as follows:

[0064] (1) An EGFR gene sequence (as shown in SEQ ID NO: 8) was amplified to obtain a PCR product. The PCR product was subjected to electrophoresis detection, a band having a size consistent with the expected size of the target sequence was cut off, and the product was recovered for ligation and transformation. The amplified PCR product above was ligated with the vector (Genscript-Lenti vector) improved in Example 1 which is linearized with ClaI/SaCII (ClaI: NEB, R0197V; SaCII: NEB, R0157S) by using GenBuilder (GenScript, M00712) to obtain a ligated product. The ligated product was transformed into JM108 (GenScript, NA) competent cells by a thermal shock method, the transformed product was evenly spread on an LB plate culture medium (GenScript, NA) containing ampicillin resistance with a spreader, and the plate was inversely placed in a 37° C. incubator for overnight culture. A single colony was picked with a sterile pipette tip as a PCR template for colony screening, and transferred to an LB liquid culture medium (GenScript, NA) containing ampicillin resistance for culture. The PCR product for bacterial detection was subjected to electrophoresis detection, and a clone having a band with the size consistent with the expected size was selected for bacterial shaking and plasmid extraction.

[0065] (2) A Cas9 gene sequence (as shown in SEQ ID NO: 9) was amplified to obtain a PCR product. The PCR product was subjected to electrophoresis detection, a band having a size consistent with the expected size of the target sequence was cut off, and the product was recovered for ligation and transformation. The amplified PCR product above was ligated with the vector (Genscript-Lenti vector) improved in Example 1 which is linearized with XhoII/XbaI (XhoI: NEB, R0146L; XbaI: NEB, R0145S) by using GenBuilder (GenScript, M00712) to obtain a ligated product. The ligated product was transformed into JM108 (GenScript, NA) competent cells by a thermal shock method, the transformed product was evenly spread on an LB plate culture medium (GenScript, NA) containing ampicillin resistance with a spreader, and the plate was inversely placed in a 37° C. incubator for overnight culture. A single colony was picked with a sterile pipette tip as a PCR template for colony screening, and transferred to an LB liquid culture medium (GenScript, NA) containing ampicillin resistance for culture. The PCR product for bacterial detection was subjected to electrophoresis detection, and a clone having a band with the size consistent with the expected size was selected for bacterial shaking and plasmid extraction.

[0066] (3) An EGFP gene sequence (as shown in SEQ ID NO: 12) was amplified to obtain a PCR product. The PCR product was subjected to electrophoresis detection, a band having a size consistent with the expected size of the target sequence was cut off, and the product was recovered for ligation and transformation. The amplified PCR product above was ligated with the vector obtained in step (2) which is linearized with BamHI (BamHI: NEB, R3136V) by using GenBuilder (GenScript, M00712) to obtain a ligated product. The ligated product was transformed into JM108 (GenScript. NA) competent cells by a thermal shock method, the transformed product was evenly spread on an LB plate culture medium (GenScript, NA) containing ampicillin resistance with a spreader, and the plate was inversely placed in a 37° C. incubator for overnight culture. A single colony was picked with a sterile pipette tip as a PCR template for colony screening, and transferred to an LB liquid culture medium (GenScript, NA) containing ampicillin resistance for culture. The PCR product for bacterial detection was subjected to electrophoresis detection, and a clone having a band with the size consistent with the expected size was selected for bacterial shaking and plasmid extraction.

[0067] (4) A CCR2 gene sequence (as shown in SEQ ID NO: 10) was amplified to obtain a PCR product. The PCR product was subjected to electrophoresis detection, a band having a size consistent with the expected size of the target sequence was cut off, and the product was recovered for ligation and transformation. The amplified PCR product above was ligated with the vector obtained in step (3) which is linearized with AfeI (AfeI: NEB, R0652L) by using GenBuilder (GenScript, M00712) to obtain a ligated product. The ligated product was transformed into JM108 (GenScript, NA) competent cells by a thermal shock method, the transformed product was evenly spread on an LB plate culture medium (GenScript, NA) containing ampicillin resistance with a spreader, and the plate was inversely placed in a 37° C. incubator for overnight culture. A single colony was picked with a sterile pipette tip as a PCR template for colony screening, and transferred to an LB liquid culture medium (GenScript, NA) containing ampicillin resistance for culture. The PCR product for bacterial detection was subjected to electrophoresis detection, and a clone having a band with the size consistent with the expected size was selected for bacterial shaking and plasmid extraction.

TABLE-US-00001 TABLE 1 Length of fragments Name of genes (including P2A and expressed by the Blasticidin) inserted to the vector MCS site EGFR 4104 bps Cas9 4698 bps Cas9 + EGFP 5349 bps CCR2 + Cas9 + 6471 bps EGFP EGFR + Cas9 8256 bps

[0068] The maps of the constructed test vectors are shown in FIGS. 2A to 2E, respectively.

2.2 Cell Preparation

[0069] 1) 24 h before lentiviral packaging, suspended 293F cells were inoculated in a 125 mL triangular flask at a density of (3.5-4)×10.sup.6 cells/mL;

[0070] 2) the cell culture flask was placed in a 37° C., 5% CO.sub.2 incubator for culture, and the rotating speed of the shaker was adjusted to 125 rpm, followed by overnight culture; and

[0071] 3) the cell viability and the number of cells were measured the next day, and when the cell viability reached 90% or above and the total number of cells reached 7×10.sup.9, the next step was performed.

2.3 Plasmid Transfection

[0072] 1) According to the experimental requirements, 125 mL shake flasks are prepared for different groups (see Table 2 for grouping), and transfection was carried out simultaneously under the same conditions;

[0073] 2) the transfection method was as follows: on the day of virus packaging, cells were observed and measured for the viability, and if the cells were bright and round and had a viability greater than 90%. the lentiviral packaging could be performed; according to the density of 3.5×10.sup.6 cells/mL, cell fluid having a volume V.sub.1 was taken; 5% V.sub.total LV-MAX™ Supplement (Gibco, A35348) was added; a (85% V.sub.total−V.sub.1) fresh culture medium (Thermo, A3583401) was added, and then transferred to a C02 incubator for use. Plasmids were taken out and thawed, and then transfection was carried out according to the following procedure: DNA diluent was prepared, to which packaging mix plasmids (Thermo, A43237) at 1.5 ug/ml relative to the total volume, then target plasmids at 1 ug/ml relative to the total volume, and then Opti-MEM with a ratio of 5% relative to the total volume were added, and the mixture was mixed uniformly and placed at room temperature. Transfection reagent diluent was prepared, to which Opti-MEM with a ratio of 5% relative to the total volume, and then an LV-MAX transfection reagent at 1.5 pg/ml relative to the total volume were added, and the mixture was mixed uniformly and incubated at room temperature for 1 min. The DNA diluent and the transfection reagent diluent were mixed uniformly, and the mixture was incubated at room temperature for 10 min, then added to a shake flask and shaken gently. The culture flask was put back into the incubator and cultured for additional 5-6 hours; after transfection, the cells were cultured at 37° C. in 5% CO2. The rotating speed of the shaker (Kylin-Bell, Camel Shaker Model TS-2) was set to be 125 rpm; 4% Voaai LV-MAX™ Enhancer (Gibco, A35348) was added after 5-6 hours, and the shake flask was put back into the incubator and continued to be cultured.

2.4 Virus Collection

[0074] 1) The virus supernatant was collected 72 h after transfection;

[0075] 2) after the virus supernatant was obtained, the supernatant was first subjected to centrifugation in a centrifuge (Eppendorf, 5810R) at 4° C. and 1000-3000 rpm for 10 min, and then transferred to a new centrifuge tube and filtered with a 0.45-micron filter membrane (Millipore. Cat. No. SLHP033RB);

[0076] 3) the obtained supernatant was mixed uniformly with a concentrated reagent (40% PEG8000, 4% sodium chloride) in a ratio of 4:1, and then left overnight at 4° C.; the next day, the concentrated virus solution was centrifuged at 4° C. and 1500 g for 45 min to 1 h; the supernatant was discarded, and an appropriate amount of 4° C. pre-cooled DPBS (Gibco, Cat. No. 14190-144) was added, the precipitate was suspended by means of gentle and full pipetting to obtain a virus suspension, which is subpackaged and stored in a −80° C. refrigerator.

2.5 Determination of Virus Titer by p24 Method

[0077] A commercial kit Lenti-X™ p24 Rapid Titer Kit (Takara, Cat. No. 632200) was used for titer determination, and 50 μL of virus suspension was taken for detection. For specific operations, reference was made to the kit instructions. A microplate reader was used for determination (the absorbance value of the sample was detected at an OD of 450 nm), and the data was imported into the template for data processing. The results are as shown in Table 2.

[0078] The calculation formula is as follows:

1 virus particle is approximately equal to 8×10.sup.−5 pg p24;
1 ng p24 is approximately equal to about 1.25×10.sup.7 viral particles:
1 IFU is approximately equal to 100 to 1000 virus particles, and the median is taken to calculate the virus titer.

TABLE-US-00002 TABLE 2 Name of Inserted fragments Titer value (× inserted (including P2A and 10{circumflex over ( )}6 genes Blasticidin) IFU/mL) EGFR 4104 bps 147 Cas9 4698 bps 135 Cas9 + EGFP 5349 bps 86 CCR2 + EGFP + 6471 bps 47 Cas9 EGFR + Cas9 8280 bps 68

[0079] The virus titer as calculated in Table 2 shows that the packaging capacity of the improved lentiviral expression vector of the present invention can exceed the upper limit (3 kb) of the traditional expression vector; and the improved expression vector can achieve a high titer for genes longer than 5 kb.

Example 3. Determination of Functional Titer of Improved Lentiviral Expression Vector

[0080] With reference to the method of Example 2.1. some of the long-fragment genes (see Table 3) were selected and subcloned into the improved lentiviral expression vector backbone. In addition, with reference to the determination method in Example 2.5. the p24 physical titer determination and the FACS functional titer test were carried out.

TABLE-US-00003 TABLE 3 Name of inserted Length of inserted genes fragments EGFR 4104 bps Cas9 4698 bps Cas9 + EGFP 5349 bps CCR2 + EGFP + 6471 bps Cas9

[0081] The experimental steps of the FACS functional test were as follows:

(1) 293T cells were plated on a 96-well plate and cultured overnight, so that the cell confluence rate fell within the range of 30% to 50% and the initial cell concentration was approximately 3×10.sup.5/mL before transfection;
2) the virus stock solution was diluted according to the virus concentration gradient (10.sup.−1 to 10.sup.−6) in Table 4;
3) the culture medium from the 96-well plate was removed, and 1 mL of the culture medium (DMEM+10% FBS) containing viruses diluted in gradients and Polybrene was added to the 96-well plate containing cells, followed by centrifugation at 800 rpm for 30 min;
4) 24 hours after the cells were infected by viruses, the culture medium was changed to a 2 mL fresh Polybrene culture medium;
5) 72 hours after the cells were infected by viruses, FACS was performed to detect the percentage of positive cells expressing enhanced green fluorescent protein (EGFP); and
6) groups with the infection rate in a range of 1% to 40% were selected for titer calculation. The calculation formula is:

virus titer=initial cell number×% EGFP×1.5/dilution factor (wherein % EGFP represents the percentage of positive cells expressing EGFP, and the dilution factor is as shown in Table 4).

TABLE-US-00004 TABLE 4 Culture medium No. Dilution factor Virus volume containing Polybrene 1 1 × 10.sup.−2 22 μL 2178 μL 2 1 × 10.sup.−3 220 μL of Tube #1 1980 μL 3 1 × 10.sup.−4 220 μL of Tube #2 1980 μL 4 1 × 10.sup.−5 220 μL of Tube #3 1980 μL 5 1 × 10.sup.−6 220 μL of Tube #4 1980 μL

[0082] The experimental results are shown in FIGS. 3A to 3C, which indicate that the improved lentiviral expression vector has good repeatability. According to the calculation results of the virus titer as shown in Table 5, from the perspective of the functional titer, if the inserted fragment exceeds 5 kb, although a high physical titer can be obtained, the functional titer will be greatly reduced. Since there is no suitable antibody for Cas9, the functional titer is not detected in this group.

TABLE-US-00005 TABLE 5 Length p24 of inserted physical FACS fragments titer functional titer Name of (+ 498 bps (× 10{circumflex over ( )}6 (× 10{circumflex over ( )}6 vectors flag&P2A&blast) IFU/mL) TU/mL) EGFR 3773 bps 343.0155 333 Cas9 4698 bps 358.608875 NA Cas9 + EGFP 5498 bps 184.65725 10.25 CCR2 + 6622 bps 261.170125 1.87 EGFP + Cas9

[0083] The sequence information involved in the present invention is as follows:

TABLE-US-00006 CMV enhancer (SEQ ID NO: 1): GACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATC AAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTAT TAGTCATCGCTATTACCATG CMV promoter (SEQ ID NO: 2): GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGC GGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGAC TTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAA TGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG CGC Truncated 5′ LTR (SEQ ID NO: 3) GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCT CTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAG CTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGT CAGTGTGGAAAATCTCTAGCA EFS promoter (SEQ ID NO: 4) TAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTC AGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTT GGGGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGG TGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGC TCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAG TGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTT GCCGCCAGAACACAGG P2A linker sequence (SEQ ID NO: 5): GGATCCGGCGCAACAAACTTCTCTCTGCTGAAACAAGCCG GAGATGTCGAAGAGAATCCTGGACCG Blasticidin resistance gene (SEQ ID NO: 6): ATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTG AAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGA AGACTACAGCGTCGCCAGCGCAGCTCTCTGTAGCGACGGC CGCATCTTCACTGGTGTCAATGTATATCATTTTACTGGGG GACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGC TGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGA AATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCC GACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCAT AGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATT CGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAA 3′ LTR sequence with a partially deleted U3 region (SEQ ID NO: 7): CTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATATCC TTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGA TTGGCAGAACTACACACCAGGGCCAGGGATCAGATATCCA CTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGC AAGAGAAGGTAGAAGAAGCCAATGAAGGAGAGAACACCCG CTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCG GAGAGAGAAGTATTAGAGTGGAGGTTTGACAGCCGCCTAG CATTTCATCACATGGCCCGAGAGCTGCATCCGGACTGTAC TGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTC TCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAA GCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTT GTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAG TCAGTGTGGAAAATCTCTAGCAG EGFR sequence (SEQ ID NO: 8): ATGCGACCCTCCGGGACGGCCGGGGCAGCGCTCCTGGCGC TGCTGGCTGCGCTCTGCCCGGCGAGTCGGGCTCTGGAGGA AAAGAAAGTTTGCCAAGGCACGAGTAACAAGCTCAGGCAG TTGGGCACTTTTGAAGATCATTTTCTCAGCCTCCAGAGGA TGTTCAATAACTGTGAGGTGGTCCTTGGGAATTTGGAATT ACCTATGTGCAGAGGAATTATGATCTTTCCTTCTTAAAGA CCATCCAGGAGGTGGCTGGTTATGTCCTCATTGCCCTCAA CACAGTGGAGCGAATTCCTTTGGAAAACCTGCAGATCATC AGAGGAAATATGTACTACGAAAATTCCTATGCCTTAGCAG TCTTATCTAACTATGATGCAAATAAAACCGGACTGAAGGA GCTGCCCATGAGAAATTTACAGGAAATCCTGCATGGCGCC GTGCGGTTCAGCAACAACCCTGCCCTGTGCAACGTGGAGA GCATCCAGTGGCGGGACATAGTCAGCAGTGACTTTCTCAG CAACATGTTGATGGACTTCCAGAACCACCTGGGCAGCTGC CAAAAGTGTGATCCAAGCTGTCCCAATGGGAGCTGCTGGG GTGCAGGAGAGGAGAACTGCCAGAAACTGACCAAAATCAT CTGTGCCCAGCAGTGCTCCGGGCGCTGCCGTGGCAAGTCC CCCAGTGACTGCTGCCACAACCAGTGTGCTGCAGGCTGCA CAGGCCCCCGGGAGAGCGACTGCCTGGTCTGCCGCAAATT CCGAGACGAAGCCACGTGCAAGGACACCTGCCCCCCACTC ATGCTCTACAACCCCACCACGTACCAGATGGATGTGAACC CCGAGGGCAAATACAGCTTTGGTGCCACCTGCGTGAAGAA GTGTCCCCGTAATTATGTGGTGACAGATCACGGCTCGTGC GTCCGAGCCTGTGGGGCCGACAGCTATGAGATGGAGGAAG ACGGCGTCCGCAAGTGTAAGAAGTGCGAAGGGCCTTGCCG CAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGAC TCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAA ACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGT GGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTG GATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAA TCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAG GACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGC GGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCG TCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAA GGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAA AATTTGTGCTATGCAAATACAATAAACTGAAAAAACTGTT TGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGA GGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATG CCTTGTGCTCCCCCGAGGGCTGCTGGGGCCGGGAGCCCAG GGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAA TGCGTGGACAAGTGGAACCTTCTGGAGGGTGAGCCAAGGG AGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGA GTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGG GGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACG GCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGG AGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGC CATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGAT GCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCC TAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTC CTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCA TGCGAAGGCGCCACATCGTTCGGAAGCGCACGCTGCGGAG GCTGCTGCAGGAGAGGGAGCTTGTGGAGCCTCTTACACCC AGTGGAGAAGCTCCCAACCAAGCTCTCTTGAGGATCTTGA AGGAAACTGAATTCAAAAAGATCAAAGTGCTGGGCTCCGG TGCGTTCGGCACGGTGTATAAGGGACTCTGGATCCCAGAA GGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAA GAGAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGA TGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTG TGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGC TCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTA TGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTG CTCAACTGGTGTGTGCAGATCGCAAAGGGCATGAACTACT TGGAGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCCAG GAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACA GATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAAG AATACCATGCAGAAGGAGGCAAAGTGCCTATCAAGTGGAT GGCATTGGAATCAATTTTACACAGAATCTATACCCACCAG AGTGATGTCTGGAGCTACGGGGTGACTGTTTGGGAGTTGA TGACCTTTCGATCCAAGCCATATGACGGAATCCCTGCCAG CGAGATCTCCTCCATCCTGGAGAAAGGAGAACGCCTCCCT CAGCCACCCATATGTACCATCGATGTCTACATGATCATGG TCAAGTGCTGGATGATAGACGCAGATAGTCGCCCAAAGTT CCGTGAGTTGATCATCGAATTCTCCAAAATGGCCCGAGAC CCCCAGCGCTACCTTGTCATTCAGGGGGATGAAAGAATGC ATTTGCCAAGTCCTACAGACTCCAACTTCTACCGTGCCCT GATGGATGAAGAAGACATGGACGACGTGGTGGATGCCGAC GAGTACCTCATCCCACAGCAGGGCTTCTTCAGCAGCCCCT CCACGTCACGGACTCCCCTCCTGAGCTCTCTGAGTGCAAC CAGCAACAATTCCACCGTGGCTTGCATTGATAGAAATGGG CTGCAAAGCTGTCCCATCAAGGAAGACAGCTTCTTGCAGC GATACAGCTCAGACCCCACAGGCGCCTTGACTGAGGACAG CATAGACGACACCTTCCTCCCAGTGCCTGAATACATAAAC CAGTCCGTTCCCAAAAGGCCCGCTGGCTCTGTGCAGAATC CTGTCTATGACAATGAGCCTCTGAACCCCGCGCCCAGCAG AGACCCACACTACCAGGACCCCCACAGCACTGCAGTGGGC AACCCCGAGTATCTCAACACTGTCCAGCCCACCTGTGTCA ACAGCACATTCGACAGCCCTGCCCACTGGGCCCAGAAAGG CAGCCACCAAATTAGCCTGGACAACCCTGACTACCAGCAG GACTTCTTTCCCAAGGAAGCCAAGCCAAATGGCATCTTTA AGGGCTCCACAGCTGAAATGCAGAATACCTAAGGGTCGCG CCACAAAGCAGTGAATTTATTGGAGCA Cas9 sequence (SEQ ID NO: 9): ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCA ACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGT GCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGG CACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCG ACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAAC CGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGC TATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGG ACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGT GGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGC AACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCA CCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGA CAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCAC ATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC TGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCA GCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCC ATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTG CCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGC CCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAAC CTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGA GCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAG CAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCC CAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCA AGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAG AGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCT ATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCC TGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTA CAAAGAGATTTTCTTGGACCAGAGCAAGAACGGCTACGCC GGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACA AGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGA GGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGG AAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGA TCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGA AGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATC GAGAAGATCCTGACCTTCCGCATCCCCTACTAGGTGGGCC CTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAG AAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAA GTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGC GGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGT GCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTG TATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAA TGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGC CATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACC GTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGT GCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTT CAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATT ATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGG ACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGA GGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCC CACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGC GGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGAT CAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTG GATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCA TGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGA CATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTG CACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTA AGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCT CGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTG ATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGAC AGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGG CATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCC GTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGT ACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCAT ATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACA ACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAG CGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAG AACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCC AGAGAAAGTTCGACAATCTGACCAAGGCGGAGAGAGGCGG CCTGAGCAACTGGATAAGGGCGGCTTCATCAAGAGACAGC TGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGAT CCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGAC AAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCA AGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAA AGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCC TACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGT ACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAA GGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAG GAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCA ACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAA CGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGC GAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTG CCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATAT CGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAA GAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCG CCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTT CGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCC AAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGA AAGAGCTACTGGGGATCACCATCATGGAAAGAAGCAGCTT CGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTAC AAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGT ACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCT GGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCC GTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCC ACTATGAGAAGCTGAAGGGCTCGCCCGAGGATAATGAGCA GAAACAGCTGTTTGTGGAACAGCACAAGGACTACCTGGAC GAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGA TCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTA CAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAG AATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCC CTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAA GAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTG ATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCG ACCTGTCTCAGCTGGGAGGCGACAAGCGACCTGCCGCCAC AAAGAAGGCTGGACAGGCTAAGAAGAAGAAA CCR2 sequence (SEQ ID NO: 10): ATGCTGTCCACATCTCGTTCTCGGTTTATCAGAAATACCA ACGAGAGCGGTGAAGAAGTCACCACCTTTTTTGATTATGA TTACGGTGCTCCCTGTCATAAATTTGACGTGAAGCAAATT GGGGCCCAACTCCTGCCTCCGCTCTACTCGCTGGTGTTCA TGTTTGGTTTTGTGGGCAACATGCTGGTCGTCCTCATCTT AATAAACTGCAAAAAGCTGAAGTGCTTGACTGACATTTAC CTGCTCAACCTGGCCATCTCTGATCTGCTTTTTCTTATTA CTCTCCCATTGTGGGCTCACTCTGCTGCAAATGAGTGGGT CTTTGGGAATGCAATGTGCAAATTATTCACAGGGCTGTAT CACATCGGTTATTTTGGCGGAATCTTCTTCATCATCCTCC TGACAATCGATAGATACCTGGCTATTGTCCATGCTGTGTT TGCTTTAAAAGCCAGGACGGTCACCTTTGGGGTGGTGACA AGTGTGATCACCTGGTTGGTGGCTGTGTTTCCTTCTGTCC GAGGAATCATGTTTACTAAATGCCAGAAAGAAGATTCTGT TTATGTCTGTGGCCCTTATTTTCCACGAGGATGGAATAAT TTCCACACAATAATGAGGAACATTTTGGGGCTGGTCCTGC CGCTGCTCATCATGGTCATCTGCTACTCGGGAATCCTGAA AACCCTGCTTCGGTGTCGAAACGAGAAGAAGAGGCATAGG GCAGTGAGAGTCATCTTCACCATCATGATTGTTTACTTTC TCTTCTGGACTCCCTATAATATTGTCATTCTCCTGAACAC CTTCCAGGAATTCTTCGGCCTGAGTAACTGTGAAAGCACC AGTCAACTGGACCAAGCCACGCAGGTGACAGAGACTCTTG GGATGACTCACTGCTGCATCAATCCCATCATCTATGCCTT CGTTGGGGAGAAGTTCAGAAGCCTTTTTCACATAGCTCTT GGCTGTAGGATTGCCCCACTCCAAAAACCAGTGTGTGGAG GTCCAGGAGTGAGACCAGGAAAGAATGGTGAAAGTGACTA CACAAGGACTCCTCGATGGTCGTGGAAAAGGAAAGTCAAT TGGCAGAGCCCCTGAAGCCAGTCTTCAGGACAAAGAAGGA GCC Flag sequence (SEQ ID NO: 11): GATTACAAAGACGATGACGATAAGGATTACAAAGACGATG ACGATAAGGATTACAAAGACGATGACGATAAG EGFP sequence (SEQ ID NO: 12): GCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGG TGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGG CCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCC ACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCG GCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGGACCACCC TGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCA CATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAA GGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACG GCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGA CACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTC AAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACA ACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCA GAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAAC ATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGC AGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGA CAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGAC CCCAACGAGAAGCGCGATCACATGGTGCTGCTGGAGTTGG TGACCGCCGCCGGGATCACTCTCGGGATGGACGAGCTGT