LOCUS       sequence-491511-        6806 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  ZFa Retrovirus  expressing VP16-ZF43-2A-TagBFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6806)
  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 6806)
  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-ZF43-2A-TagBFP-WPRE_pKG2988
FEATURES             Location/Qualifiers
     source          1..6806
                     /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(2218..2919)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             complement(2932..2988)
                     /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(3541..3561)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3568..3633)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      3633..3642
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3709..3733)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3776..4150
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     complement(3822..3838)
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             complement(4160..4576)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(4176..4192)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(4208..4230)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    4635..4992
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     LTR             5055..5648
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     promoter        5678..5782
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5783..5851,5852..6643)
                     /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(6001..6020)
                     /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 attaagcttg tgccccagtt tgctagggag gtcgcagtat ctggccactg ccacctcgtg
     2281 ctgctcgacg tacgtttcgt tgttggcctc cttgattctt tccagtctgt agtccacata
     2341 gtagacgcca ggcatcttga ggttcttagc gggtttcttg gatctatatg tggtcttgat
     2401 gtttgcgatc agatggctcc cgcccacgag cttcagggcc atgtcgtttc tgccttccag
     2461 gccgccgtca gcggggtaca gcgtctcggt gaaggcctcc cagccgagtg ttttcttctg
     2521 catcacaggg ccgttggatg tgaagttcac ccctctgatc ttgacgttgt agatgaggca
     2581 gccgtcctgg aggctggtgt cctgggtagc ggtcagcacg cccccgtctt cgtatgtggt
     2641 gactctctcc catgtgaagc cctcagggaa ggactgcttg aagaagtcgg ggatgccctg
     2701 ggtgtggttg atgaaggtct tgctgccgta gaggaagcta gtagccagga tgtcgaaggc
     2761 gaaggggaga gggccgccct cgaccacctt gattctcatg gtctgggtgc cctcgtaggg
     2821 cttgccttcg ccctcggatg tgcacttgaa gtgatggttg tccacggtgc cctccatgta
     2881 cagcttcatg tgcatgttct ccttaatcag ctcgctcata cttcctggtg caggtccagg
     2941 gttctcctcc acgtctccag cctgcttcag caggctgaag ttagtagctc cgcttccaga
     3001 tcctctcagg tgggttttta ggtgccggtt caggttgtcg cggcgactga agttgcgcat
     3061 gcatattcgg cattggaatg gcttctcgcc ggtgtgcgta cgtagatgcc ccgccaagtg
     3121 ctccttctgg gagaaatttc gcatacagat ccgacactga aacggttttt caccggtatg
     3181 agtacgggta tgcctgtcaa gcctgtcctg gcgcgaaaag ttccgcatgc aaatgcgaca
     3241 ctggaagggg cgctccccgg gtctagctcc accaccgtat tcgtctatac ccaaggcatc
     3301 agtaaacatc tgttcaaatt cgaaatcggc catgtccaaa gcaccgtatg gagctgaatc
     3361 gtgtggtgtg aaaccaggtc ctggtgaatc tccatctcct aacatatcca agtcaaaatc
     3421 gtctaaggca tctgcatgtg ccatagctac gtcctcaccg tccaaatgta actcatcacc
     3481 taaagaaaca tcggttggtg gggcctgcag tccaccgtcg acgggaacgc catggatacc
     3541 gactttgcgc tttttcttag gagccatttt atcgtcatcg tctttgtagt caatatcgtg
     3601 gtccttgtag tcgccatcgt gatccttata atccatggtg gcacattggg atccaagctt
     3661 gaattcagta gtaccggatc cagtcgactg aattggttcc catggtggag cctgcttttt
     3721 tgtacaaact tgtttgatag cttatcgata ccgtcgatta actagctaga tccggcagtc
     3781 tagaggatgg tccacccccg gggtcggcag ccttcacgtg ggcggcgtgt atccaagctg
     3841 cgatgccgtc tactttgagg gcggtggggg tggtcagcag gactgtgtaa ggtcctttcc
     3901 agcgaggttc taggttctta gtctggtgtc ggcggaccca cactgtgtcg ccgactcggt
     3961 aagggtgagg taccaccggt cggtccagtt gttcttggta ggctgccgcc agaggtctcc
     4021 agacttcgtg ctggactaag tagagagcct gtaagtgagc ttggagagag gggctgttag
     4081 taactcttgt catgtcaggg tcagggaagt ttacaagggg cgggggtgcc ccatataaga
     4141 tctcatatgg ccatatgggg gcgcctagag aaggagtgag ggctggataa agggaggatc
     4201 gaggcggggt cgaacgagga ggttcaaggg ggagagacgg ggcggatgga ggaagaggag
     4261 gcggaggctt agggtgtaca aagggcttga cccagggagg ggggtcaaaa gccaaggctt
     4321 cccaggtcac gatgtagggg acctggtctg ggtgtccatg cgggccaggt gaaaagacct
     4381 tgatcttaac ctgggtgatg aggtctcggt taaaggtgcc gtctcgcggc catccgacgt
     4441 taaaggttgg ccattctgca gagcagaagg taacccaacg tctcttcttg acatctaccg
     4501 actggttgtg agcgatccgc tcgacatctt tccagtgacc taaggtcaaa cttaagggag
     4561 tggtaacagt ctggccctaa ttttcagaca aatacagaaa cacagtcaga cagagacaac
     4621 acagaacgat gctgcagcag acaagacgcg cggcgcggct tcggtcccaa accgaaagca
     4681 aaaattcaga cggaggcggg aactgtttta ggttctcgtc tcctaccaga accacatatc
     4741 cctcctatta ggggggtgca ccaaagagtc caaaacgatc gggatttttg gactcaggtc
     4801 gggccacaaa aacggccccc gaagtccctg ggacgtctcc cagggttgcg gccgggtgtt
     4861 ccgaactcgt cagttccacc acgggtccgc cagatacaga gctagttagc taactagtac
     4921 cgacgcaggc gcataaaatc agtcatagac actagacaat cggacagaca cagataagtt
     4981 gctggccagc ttacctcccg gtggtgggtc ggtggtccct gggcaggggt ctcccgatcc
     5041 cggacgagcc cccaaatgaa agacccccgc tgacgggtag tcaatcactc agaggagacc
     5101 ctcccaagga acagcgagac cacaagtcgg atgcaactgc aagagggttt attggataca
     5161 cgggtacccg ggcgactcag tcaatcggag gactggcgcg ccgagtgagg ggttgtgggc
     5221 tcttttattg agctcgggga gcagaagcgc gcgaacagaa gcgagaagcg aactgattgg
     5281 ttagttcaaa taaggcacag ggtcatttca ggtccttggg gcaccctgga aacatctgat
     5341 ggttctctag aaactgctga gggctggacc gcatctgggg accatctgtt cttggccctg
     5401 agccggggca ggaactgctt accacagata tcctgtttgg cccatattca gctgttccat
     5461 ctgttcttgg ccctgagccg gggcaggaac tgcttaccac agatatcctg tttggcccat
     5521 attcagctgt tccatctgtt cctgaccttg atctgaactt ttctattctc agttatgtat
     5581 ttttccatgc cttgcaaaat ggcgttactt aagctagctt gccaaaccta caggtggggt
     5641 ctttcattat gcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5701 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5761 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5821 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5881 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5941 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     6001 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     6061 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     6121 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     6181 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6241 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6301 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6361 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6421 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6481 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6541 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6601 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6661 ctcatatata ctttagattg atttgcggcc ggccgcaaaa ggatctaggt gaagatcctt
     6721 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6781 cccgtagaaa agatcaaagg atcttc
//
