LOCUS       sequence-491514-        6848 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  ZFa Retrovirus expressing VP16-ZF37-2A-mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6848)
  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 6848)
  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: pMXs-VP16-ZF37-2A-mCherry-WPRE_pKG2987
FEATURES             Location/Qualifiers
     source          1..6848
                     /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"
     LTR             890..1359
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    1539..2127
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1645..1656
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2054..2074
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    2144..2168
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(2221..2241)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2248..2955)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2523..2542)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2792..2810
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(2968..3024)
                     /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             complement(3577..3597)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3604..3669)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      3669..3678
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3751..3775)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3818..4192
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     complement(3864..3880)
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             complement(4202..4618)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(4218..4234)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(4250..4272)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    4677..5034
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     LTR             5097..5690
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     promoter        5720..5824
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5825..5893,5894..6685)
                     /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(6043..6062)
                     /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 tgcaactatg gccatttaat
      841 gtaaatactt aagaaaaaaa accaaattaa ttttgataca tgctgcatgt gaaagacccc
      901 cgctgacggg tagtcaatca ctcagaggag accctcccaa ggaacagcga gaccacaagt
      961 cggatgcaac tgcaagaggg tttattggat acacgggtac ccgggcgact cagtcaatcg
     1021 gaggactggc gccccgagtg aggggttgtg ggctctttta ttgagctcgg ggagcagaag
     1081 cgcgcgaaca gaagcgagaa gcgaactgat tggttagttc aaataaggca cagggtcatt
     1141 tcaggtcctt ggggcaccct ggaaacatct gatggttctc tagaaactgc tgagggctgg
     1201 accgcatctg gggaccatct gttcttggcc ctgagccggg gcaggaactg cttaccacag
     1261 atatcctgtt tggcccatat tcagctgttc catctgttct tggccctgag ccggggcagg
     1321 aactgcttac cacagatatc ctgtttggcc catattcagc tgttccatct gttcctgacc
     1381 ttgatctgaa cttctctatt ctcagttatg tatttttcca tgccttgcaa aatggcgtta
     1441 cttaagctag cttgccaaac ctacaggtgg ggtctttcat tccccccttt ttctggagac
     1501 taaataaaat cttttatttt atcgtcgacg gtatcgatgc ggggaggcgg cccaaaggga
     1561 gatccgactc gtctgagggc gaaggcgaag acgcggaaga ggccgcagag ccggcagcag
     1621 gccgcgggaa ggaaggtccg ctggattgag ggccgaaggg acgtagcaga aggacgtccc
     1681 gcgcagaatc caggtggcaa cacaggcgag cagccaagga aaggacgatg atttccccga
     1741 caacaccacg gaattgtcag tgcccaacag ccgagcccct gtccagcagc gggcaaggca
     1801 ggcggcgatg agttccgccg tggcaatagg gagggggaaa gcgaaagtcc cggaaaggag
     1861 ctgacaggtg gtggcaatgc cccaaccagt gggggttgcg tcagcaaaca cagtgcacac
     1921 cacgccacgt tgcctgacaa cgggccacaa ctcctcataa agagacagca accaggattt
     1981 atacaaggag gagaaaatga aagccatacg ggaagcaata gcatgataca aaggcattaa
     2041 agcagcgtat ccacatagcg taaaaggagc aacatagtta agaataccag tcaatctttc
     2101 acaaattttg taatccagag gttgattatc gataagctat cgaaccactt tgtacaagaa
     2161 agctgggtct agatatcagc gtaatacggt tatccacaga atcaggggat aacgttgtta
     2221 tacctttctc ttcttttttg gcaattgctt gtacagctcg tccatgccgc cggtggagtg
     2281 gcggccctcg gcgcgttcgt actgttccac gatggtgtag tcctcgttgt gggaggtgat
     2341 gtccaacttg atgttgacgt tgtaggcgcc gggcagctgc acgggcttct tggccttgta
     2401 ggtggtcttg acctcagcgt cgtagtggcc gccgtccttc agcttcagcc tctgcttgat
     2461 ctcgcccttc agggcgccgt cctcggggta catccgctcg gaggaggcct cccagcccat
     2521 ggtcttcttc tgcattacgg ggccgtcgga ggggaagttg gtgccgcgca gcttcacctt
     2581 gtagatgaac tcgccgtcct gcagggagga gtcctgggtc acggtcacca cgccgccgtc
     2641 ctcgaagttc atcacgcgct cccacttgaa gccctcgggg aaggacagct tcaagtagtc
     2701 ggggatgtcg gcggggtgct tcacgtaggc cttggagccg tacatgaact gaggggacag
     2761 gatgtcccag gcgaagggca gggggccacc cttggtcacc ttcagcttgg cggtctgggt
     2821 gccctcgtag gggcggccct cgccctcgcc ctcgatctcg aactcgtggc cgttcacgga
     2881 gccctccatg tgcaccttga agcgcatgaa ctccttgatg atggccatgt tatcctcctc
     2941 gcccttgctc accatacttc ctggtgcagg tccagggttc tcctccacgt ctccagcctg
     3001 cttcagcagg ctgaagttag tagctccgct tccagatcct ctcaggtggg tttttaggtg
     3061 tctagtcaat tgatcatgtc tactgaagtt gcgcatgcat attcggcatt ggaatggctt
     3121 ctcgccggtg tgcgtacgca gatgcctcct caaattagct ctatcggaga aatttcgcat
     3181 acagatccga cactgaaacg gtttttcacc ggtatgagta cgggtatgtc tttgcaaaat
     3241 gaaatttctc gaaaagttcc gcatgcaaat gcgacactgg aaggggcgct ccccgggtct
     3301 agctccacca ccgtattcgt ctatacccaa ggcatcagta aacatctgtt caaattcgaa
     3361 atcggccatg tccaaagcac cgtatggagc tgaatcgtgt ggtgtgaaac caggtcctgg
     3421 tgaatctcca tctcctaaca tatccaagtc aaaatcgtct aaggcatctg catgtgccat
     3481 agctacgtcc tcaccgtcca aatgtaactc atcacctaaa gaaacatcgg ttggtggggc
     3541 ctgcagtcca ccgtcgacgg gaacgccatg gataccgact ttgcgctttt tcttaggagc
     3601 cattttatcg tcatcgtctt tgtagtcaat atcgtggtcc ttgtagtcgc catcgtgatc
     3661 cttataatcc atggtggcgg tggctcattg ggatccaagc ttgaattcag tagtaccgga
     3721 tccagtcgac tgaattggtt cccatggtgg agcctgcttt tttgtacaaa cttgtttgat
     3781 agcttatcga taccgtcgat taactagcta gatccggcag tctagaggat ggtccacccc
     3841 cggggtcggc agccttcacg tgggcggcgt gtatccaagc tgcgatgccg tctactttga
     3901 gggcggtggg ggtggtcagc aggactgtgt aaggtccttt ccagcgaggt tctaggttct
     3961 tagtctggtg tcggcggacc cacactgtgt cgccgactcg gtaagggtga ggtaccaccg
     4021 gtcggtccag ttgttcttgg taggctgccg ccagaggtct ccagacttcg tgctggacta
     4081 agtagagagc ctgtaagtga gcttggagag aggggctgtt agtaactctt gtcatgtcag
     4141 ggtcagggaa gtttacaagg ggcgggggtg ccccatataa gatctcatat ggccatatgg
     4201 gggcgcctag agaaggagtg agggctggat aaagggagga tcgaggcggg gtcgaacgag
     4261 gaggttcaag ggggagagac ggggcggatg gaggaagagg aggcggaggc ttagggtgta
     4321 caaagggctt gacccaggga ggggggtcaa aagccaaggc ttcccaggtc acgatgtagg
     4381 ggacctggtc tgggtgtcca tgcgggccag gtgaaaagac cttgatctta acctgggtga
     4441 tgaggtctcg gttaaaggtg ccgtctcgcg gccatccgac gttaaaggtt ggccattctg
     4501 cagagcagaa ggtaacccaa cgtctcttct tgacatctac cgactggttg tgagcgatcc
     4561 gctcgacatc tttccagtga cctaaggtca aacttaaggg agtggtaaca gtctggccct
     4621 aattttcaga caaatacaga aacacagtca gacagagaca acacagaacg atgctgcagc
     4681 agacaagacg cgcggcgcgg cttcggtccc aaaccgaaag caaaaattca gacggaggcg
     4741 ggaactgttt taggttctcg tctcctacca gaaccacata tccctcctat taggggggtg
     4801 caccaaagag tccaaaacga tcgggatttt tggactcagg tcgggccaca aaaacggccc
     4861 ccgaagtccc tgggacgtct cccagggttg cggccgggtg ttccgaactc gtcagttcca
     4921 ccacgggtcc gccagataca gagctagtta gctaactagt accgacgcag gcgcataaaa
     4981 tcagtcatag acactagaca atcggacaga cacagataag ttgctggcca gcttacctcc
     5041 cggtggtggg tcggtggtcc ctgggcaggg gtctcccgat cccggacgag cccccaaatg
     5101 aaagaccccc gctgacgggt agtcaatcac tcagaggaga ccctcccaag gaacagcgag
     5161 accacaagtc ggatgcaact gcaagagggt ttattggata cacgggtacc cgggcgactc
     5221 agtcaatcgg aggactggcg cgccgagtga ggggttgtgg gctcttttat tgagctcggg
     5281 gagcagaagc gcgcgaacag aagcgagaag cgaactgatt ggttagttca aataaggcac
     5341 agggtcattt caggtccttg gggcaccctg gaaacatctg atggttctct agaaactgct
     5401 gagggctgga ccgcatctgg ggaccatctg ttcttggccc tgagccgggg caggaactgc
     5461 ttaccacaga tatcctgttt ggcccatatt cagctgttcc atctgttctt ggccctgagc
     5521 cggggcagga actgcttacc acagatatcc tgtttggccc atattcagct gttccatctg
     5581 ttcctgacct tgatctgaac ttttctattc tcagttatgt atttttccat gccttgcaaa
     5641 atggcgttac ttaagctagc ttgccaaacc tacaggtggg gtctttcatt atgcaggtgg
     5701 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5761 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5821 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5881 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5941 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     6001 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     6061 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     6121 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     6181 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6241 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6301 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6361 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6421 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6481 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6541 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6601 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6661 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6721 tgatttgcgg ccggccgcaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6781 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6841 ggatcttc
//
