LOCUS       sequence-491515-        8075 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  ZFa Lentivirus expressing VP16-ZF37-2A-TagBFP from the hPGK 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8075)
  AUTHORS   Kabaria SR, Bae Y, Ehmann ME, Lende-Dorn BA, Beitz AM, Peterman EL, 
            Love KS, Ploessl DS, Galloway KE
  TITLE     Programmable promoter editing for precise control of transgene 
            expression.
  JOURNAL   Nat Biotechnol. 2025 Oct 13. doi: 10.1038/s41587-025-02854-y.
  PUBMED    41083901
REFERENCE   2  (bases 1 to 8075)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2025 Oct 13. doi: 10.1038/s41587-025-02854-y."
COMMENT     Sequence Label: LentiX_PGK-VP16-ZF37-2A-TagBFP-WPRE_pKG3958
FEATURES             Location/Qualifiers
     source          1..8075
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1058..1284
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1285..1465
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1512..1637
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2130..2363
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2548..2592
                     /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"
     misc_feature    2859..2975
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3012..3522
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3441..3459
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      3536..3545
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3545..3610
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3617..3637
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4190..4246
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4259..4960
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    4971..5559
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5024..5044)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5442..5453)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5651..5884
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5956..6077
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5993..6012)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6047..6066
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6117..6252
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6272..6291)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6273..6291)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6301..6318)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6301..6317)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6310..6332)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6459..6914
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6546..6565)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6756..6777
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6940..7044
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7045..7113,7114..7905)
                     /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(7263..7282)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
     1081 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
     1141 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
     1201 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
     1261 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
     1321 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     1381 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1441 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     1501 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1561 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1621 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1681 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1741 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1801 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1861 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1921 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1981 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     2041 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     2101 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     2161 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     2221 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     2281 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     2341 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     2401 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     2461 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     2521 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     2581 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     2641 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     2701 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     2761 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     2821 attcgattag tgaacggatc tcgacggtat cggttaactt ttaaaagaaa aggggggatt
     2881 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     2941 gaattacaaa aacaaattac aaaattcaaa attttatcgt ccagtgtggt ggctacttac
     3001 tataaggatc cggggttggg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga
     3061 cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg atctcgcaca
     3121 ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg
     3181 gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg
     3241 acgtgacaaa cggaagccgc acgtctcact agtaccctcg cagacggaca gcgccaggga
     3301 gcaatggcag cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcagggcgc
     3361 gccgagagca gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg
     3421 gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca
     3481 gtcggctccc tcgttgaccg aatcaccgac ctctctcccc agaaatggag ccaccgccac
     3541 catggattat aaggatcacg atggcgacta caaggaccac gatattgact acaaagacga
     3601 tgacgataaa atggctccta agaaaaagcg caaagtcggt atccatggcg ttcccgtcga
     3661 cggtggactg caggccccac caaccgatgt ttctttaggt gatgagttac atttggacgg
     3721 tgaggacgta gctatggcac atgcagatgc cttagacgat tttgacttgg atatgttagg
     3781 agatggagat tcaccaggac ctggtttcac accacacgat tcagctccat acggtgcttt
     3841 ggacatggcc gatttcgaat ttgaacagat gtttactgat gccttgggta tagacgaata
     3901 cggtggtgga gctagacccg gggagcgccc cttccagtgt cgcatttgca tgcggaactt
     3961 ttcgagaaat ttcattttgc aaagacatac ccgtactcat accggtgaaa aaccgtttca
     4021 gtgtcggatc tgtatgcgaa atttctccga tagagctaat ttgaggaggc atctgcgtac
     4081 gcacaccggc gagaagccat tccaatgccg aatatgcatg cgcaacttca gtagacatga
     4141 tcaattgact agacacctaa aaacccacct gagaggatct ggaagcggag ctactaactt
     4201 cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctgcac caggaagtat
     4261 gagcgagctg attaaggaga acatgcacat gaagctgtac atggagggca ccgtggacaa
     4321 ccatcacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat
     4381 gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctactag
     4441 cttcctctac ggcagcaaga ccttcatcaa ccacacccag ggcatccccg acttcttcaa
     4501 gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt
     4561 gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat
     4621 cagaggggtg aacttcacat ccaacggccc tgtgatgcag aagaaaacac tcggctggga
     4681 ggccttcacc gagacgctgt accccgctga cggcggcctg gaaggcagaa acgacatggc
     4741 cctgaagctc gtgggcggga gccatctgat cgcaaacatc aagaccacat atagatccaa
     4801 gaaacccgct aagaacctca agatgcctgg cgtctactat gtggactaca gactggaaag
     4861 aatcaaggag gccaacaacg aaacgtacgt cgagcagcac gaggtggcag tggccagata
     4921 ctgcgacctc cctagcaaac tggggcacaa gcttaattaa caacgccgat aatcaacctc
     4981 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     5041 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     5101 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     5161 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     5221 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     5281 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     5341 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     5401 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     5461 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     5521 ctcagacgag tcggatctcc ctttgggccg cctccccgcc gctcgctgat atagggcccg
     5581 gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     5641 aaggggggac tggaagggct agctcactcc caacgaagac aagatctgct ttttgcttgt
     5701 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     5761 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     5821 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     5881 agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     5941 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     6001 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     6061 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc
     6121 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     6181 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     6241 tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     6301 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     6361 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     6421 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     6481 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     6541 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     6601 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     6661 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     6721 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     6781 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     6841 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     6901 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     6961 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     7021 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     7081 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     7141 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     7201 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     7261 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     7321 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     7381 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     7441 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     7501 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     7561 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     7621 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     7681 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     7741 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     7801 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     7861 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     7921 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     7981 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     8041 gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
