LOCUS       sequence-377689-        7185 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a sgRNA for 5' tagging to VIM and contains a cassette with
            eGFP without homology arms.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7185)
  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 7185)
  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'eGFP/sgVIM
FEATURES             Location/Qualifiers
     source          1..7185
                     /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(2295..2297,2298..2300,2301..3011)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2295..2297,2298..2300,2301..3011)
                     /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(2340..2361)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2601..2620)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2948..2969
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3042..3630
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3095..3115)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3513..3524)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3702..3935
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4013..4134
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4050..4069)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4104..4123
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4174..4309
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4329..4348)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4330..4348)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4358..4375)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4358..4374)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4367..4389)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4516..4971
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4603..4622)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4813..4834
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4997..5101
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5102..5170,5171..5962)
                     /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(5320..5339)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6133..6721
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6622..6641
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6875..6892
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7009..7030
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7045..7050,7051..7068,7069..7075)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7083..7099
                     /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     7088..7110
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7107..7123
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7107..7123
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7142..7162
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7144..7162
                     /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 tgcgatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     2341 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     2401 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     2461 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     2521 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     2581 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     2641 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     2701 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     2761 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     2821 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     2881 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     2941 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     3001 agctgtacaa gatcagatct cgagccaccg gttaagtcga caatcaacct ctggattaca
     3061 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3121 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3181 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3241 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3301 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3361 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3421 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     3481 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     3541 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     3601 gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc
     3661 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     3721 ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg
     3781 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     3841 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     3901 cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt catgtcatct
     3961 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt
     4021 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4081 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4141 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4201 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4261 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac
     4321 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     4381 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     4441 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     4501 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4561 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4621 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4681 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     4741 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4801 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     4861 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4921 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     4981 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5041 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5101 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5161 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5221 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5281 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5341 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5401 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5461 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5521 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5581 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5641 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5701 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5761 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5821 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5881 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5941 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6001 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6061 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6121 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6181 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6241 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6301 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6361 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6421 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6481 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6541 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6601 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6661 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6721 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6781 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     6841 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     6901 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     6961 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     7021 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     7081 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     7141 cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagc
//
