LOCUS       sequence-377691-        7922 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a sgRNA for 5' tagging to VIM and contains a cassette with
            eGFP flanked by homology arms for VIM.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7922)
  AUTHORS   Thomsen EA, Skipper KA, Andersen S, Haslund D, Skov TW, Mikkelsen JG
  TITLE     CRISPR-Cas9-directed gene tagging using a single integrase-defective
            lentiviral vector carrying a transposase-based Cas9 off switch.
  JOURNAL   Mol Ther Nucleic Acids. 2022 Aug 4;29:563-576. doi: 
            10.1016/j.omtn.2022.08.005. eCollection 2022 Sep 13.
  PUBMED    36090759
REFERENCE   2  (bases 1 to 7922)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Ther 
            Nucleic Acids."; date: "2022-08-4"; volume: "29:563-576. doi"; 
            pages: " 10.1016/j.omtn.2022.08.005. eCollection 2022 Sep 13"
COMMENT     Sequence Label: pLV.5'VIM-eGFP-KI
FEATURES             Location/Qualifiers
     source          1..7922
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        1717..1965
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1986..2061
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2117..2234
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             join(2666..2668,2669..2671,2672..3382)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2666..2668,2669..2671,2672..3382)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2711..2732)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2972..2991)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3319..3340
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3779..4367
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3832..3852)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4250..4261)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4439..4672
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4750..4871
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4787..4806)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4841..4860
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4911..5046
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5066..5085)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5067..5085)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5095..5112)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5095..5111)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5104..5126)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5253..5708
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5340..5359)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5550..5571
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5734..5838
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5839..5907,5908..6699)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6057..6076)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6870..7458
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7359..7378
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7612..7629
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7746..7767
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7782..7787,7788..7805,7806..7812)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7820..7836
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7825..7847
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7844..7860
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7844..7860
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7879..7899
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7881..7899
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggacc tggtggacat
     1981 ggctggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     2041 aaagtggcac cgagtcggtg cttttttaag cttggcgtaa ctagatcttg agacaaatgg
     2101 cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa
     2161 gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa
     2221 aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg cgccggctcg
     2281 agggggccaa tgcgcggaaa gcccccaaaa gtcccagccc agcgctgaag taacgggacc
     2341 atgcccagtc ccaggccccg gagcaggaag gctcgagggc gcccccaccc cacccgccca
     2401 ccctccccgc ttctcgctag gtccctattg gctggcgcgc tccgcggctg ggatggcagt
     2461 gggaggggac cctctttcct aacggggtta taaaaacagc gccctcggcg gggtccagtc
     2521 ctctgccact ctcgctccga ggtccccgcg ccagagacgc agccgcgctc ccaccaccca
     2581 cacccaccgc gccctcgttc gcctcttctc cgggagccag tccgcgccac cgccgccgcc
     2641 caggccatcg ccaccctccg cagccatggt gagcaagggc gaggagctgt tcaccggggt
     2701 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2761 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2821 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     2881 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2941 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     3001 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     3061 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     3121 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     3181 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     3241 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     3301 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     3361 cggcatggac gagctgtaca agatcagatc tcgagccacc tccaccaggt ccgtgtcctc
     3421 gtcctcctac cgcaggatgt tcggcggccc gggcaccgcg agccggccga gctccagccg
     3481 gagctacgtg actacgtcca cccgcaccta cagcctgggc agcgcgctgc gccccagcac
     3541 cagccgcagc ctctacgcct cgtccccggg cggcgtgtat gccacgcgct cctctgccgt
     3601 gcgcctgcgg agcagcgtgc ccggggtgcg gctcctgcag gactcggtgg acttctcgct
     3661 ggccgacgcc atcaacaccg agttcaagaa cacccgcacc aacgagaagg tggagctgca
     3721 ggagctgaat gaccgcttcg ccaactacat cgacaaggtg cgcttccttt aagtcgacaa
     3781 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3841 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3901 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     3961 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     4021 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     4081 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     4141 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     4201 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     4261 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     4321 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga
     4381 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     4441 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     4501 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     4561 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     4621 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat
     4681 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
     4741 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4801 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4861 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc
     4921 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4981 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     5041 gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     5101 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     5161 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     5221 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     5281 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     5341 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     5401 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5461 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5521 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5581 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     5641 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     5701 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5761 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5821 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5881 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5941 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6001 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6061 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6121 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6181 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6241 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6301 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6361 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6421 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6481 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6541 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6601 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6661 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6721 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6781 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6841 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6901 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6961 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     7021 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7081 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7141 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7201 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7261 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7321 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7381 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7441 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7501 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     7561 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     7621 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     7681 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     7741 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     7801 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     7861 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa
     7921 gc
//
