LOCUS       sequence-378310-        5733 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Cp36 recombinase from Ef1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5733)
  AUTHORS   Durrant MG, Fanton A, Tycko J, Hinks M, Chandrasekaran SS, Perry NT,
            Schaepe J, Du PP, Lotfy P, Bassik MC, Bintu L, Bhatt AS, Hsu PD
  TITLE     Systematic discovery of recombinases for efficient integration of 
            large DNA sequences into the human genome.
  JOURNAL   Nat Biotechnol. 2022 Oct 10. pii: 10.1038/s41587-022-01494-w. doi: 
            10.1038/s41587-022-01494-w.
  PUBMED    36217031
REFERENCE   2  (bases 1 to 5733)
  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: "Nat 
            Biotechnol. 2022 Oct 10. pii: 10.1038/s41587-022-01494-w. doi: 
            10.1038/s41587-022-01494-w."
COMMENT     Sequence Label: pAF0084 Ef1a-Cp36-2a-EGFP
FEATURES             Location/Qualifiers
     source          1..5733
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(100..168,169..960)
                     /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(318..337)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1131..1719
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1620..1639
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1873..1890
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1935..3113
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2166..3104
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3061..3081
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4773..4826
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(4827..4829,4830..5543)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4827..4829,4830..5540)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4869..4890)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5130..5149)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5477..5498
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5575..5696
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5612..5631)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5666..5685
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        join(5728..5733,1..99)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
       61 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
      121 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
      181 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
      241 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
      301 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
      361 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
      421 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
      481 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
      541 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
      601 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
      661 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
      721 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
      781 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
      841 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
      901 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
      961 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     1021 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     1081 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     1141 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     1201 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     1261 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     1321 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     1381 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     1441 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1501 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1561 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1621 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     1681 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     1741 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     1801 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     1861 acgaccgagc gcagcgagtc agtgagcgag gaagggaaga gcggaccttc taggtcttga
     1921 aaggagtggg aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
     1981 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
     2041 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
     2101 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
     2161 cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt
     2221 gcgtgccttg aattacttcc acctggctgc agtacgtgat tcttgatccc gagcttcggg
     2281 ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt
     2341 gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg
     2401 cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga
     2461 cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt
     2521 cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga
     2581 ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc
     2641 ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg
     2701 cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga
     2761 gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga
     2821 aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt
     2881 ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg
     2941 ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt
     3001 ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc
     3061 tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgacgtacgg
     3121 ccaccatgaa gcagctgaac atccagagca gcagcaagat cacagccctg tactgcagac
     3181 tgagccggga cgatgaactg cagggcacca gcaacagcat cctgaaccag aaaatgatgc
     3241 tcgagaagta cgcccgggac aacaacttca ccaacctgga attcttcatc gacgacggct
     3301 acagcggcac caacttcaac agacccgact ggtccagact gcagagcctg atcgatgagg
     3361 gcaagatcgg ctgcatcatc gtgaaggaca tgagccggct gggcagagac tatctgagag
     3421 tgggctacta caccgacatc gtgttccccg aggccgacat ccggtttatc gccatcaaca
     3481 acggcatcga cagcaacgag agcaccgaga acgatctgac ccctttcatc aacatcatca
     3541 acgagttcta cgccaaggac accagcaaga aaatccgggc cgtgttcaag gccaagggcg
     3601 agtctggaaa gcccctggcc acaattcctc catacggcta cctgaaggac aaagaggaca
     3661 agtacaaatg ggttatcgac gaagaggcca gcaaggtcgt gaagaaaatc ttccagctgt
     3721 gcgtgcaagg ctacggccct tctcagattg ccagcgagct gatcaaagag ggcatcccca
     3781 cacctaccga gcacttcgat aagctgggca tcaacgtgtc ctctccactg agcgagatca
     3841 aaggcaactg gcagcccaag accatcagcc tgatcctgga aaagatggaa tacctgggcc
     3901 acaccgtgaa cttcaagacc tacaagaagt cctacaagag caagaagaag ctcgagaacc
     3961 ccaaagagaa gtggcagatc ttcgagaaca cccacgaggc catcatcgac caagagacat
     4021 tcgatatcgt gcagcggatc cggcagggca gaagagtgcg aaacaatctg ggcgagatgc
     4081 ccgctctgag cggcatgctg tattgtgccg attgtggcgc caagctgtac caagtgcgcg
     4141 gcaaaggatg ggagcacgag aaagaatact tcgtgtgcgc cagctaccgg aagcacaagg
     4201 gcctgtgtac aagccaccag atcaagaacg tgcaggtcga ggaactgctg ctgcacgagc
     4261 tgaagaagat caccgagtac gccagacagt acgaggacga cttcgtgaag ctggtgcaga
     4321 gcaagaccca gaatgagctg aacaagagcc tgaaagagtc caagaaagac ctggtgcacg
     4381 tgaaagagcg gatcaacaag ctggacacca tcatccagcg gctgtacgag gatatggtgg
     4441 aaggcaagct gagcgaggac cggttccaga agctgagcag caactacgag acagagcagt
     4501 gcgagctgga aaaaaaggcc accatgctcg aaaagatcat ccacgacacc gagcagacaa
     4561 ccctgaacac caccgccttt ctgaagcaag tgcgggaaca caccaccatt aacaagctga
     4621 cccctgagat catccgcatg ttcgtggaca agatcatcgt ggaaaagccc gagaagatcg
     4681 aaggcacccg gaccaagaag cagaccatct ggatctactg gaactacatc ggcatcctgg
     4741 acatcgagaa aaccgccgct agcggcagtg gagagggcag aggaagtctg ctaacatgcg
     4801 gtgacgtcga ggagaatcct ggcccagtga gcaagggcga ggagctgttc accggggtgg
     4861 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     4921 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     4981 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     5041 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     5101 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     5161 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     5221 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     5281 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     5341 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     5401 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     5461 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     5521 gcatggacga gctgtacaag taagaattcg atatcaagct tatcgataat caacaacttg
     5581 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     5641 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttagacg
     5701 tcaggtggca cttttcgggg aaatgtgcgc gga
//
