LOCUS       sequence-378304-        5658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Dn29 recombinase from Ef1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5658)
  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 5658)
  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: pAF0432 Ef1a-Dn29-2a-EGFP
FEATURES             Location/Qualifiers
     source          1..5658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             16..69
                     /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(70..72,73..786)
                     /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(70..72,73..783)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(112..133)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(373..392)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     720..741
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    818..939
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(855..874)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     909..928
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        971..1075
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1076..1144,1145..1936)
                     /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(1294..1313)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2107..2695
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2596..2615
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2849..2866
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        2911..4089
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3142..4080
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4037..4057
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4096..4105
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 gctagcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat
       61 cctggcccag tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      121 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      181 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
      241 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
      301 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
      361 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
      421 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
      481 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
      541 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
      601 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
      661 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
      721 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
      781 aagtaagaat tcgatatcaa gcttatcgat aatcaacaac ttgtttattg cagcttataa
      841 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
      901 ttctagttgt ggtttgtcca aactcatcaa tgtatcttag acgtcaggtg gcacttttcg
      961 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1021 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1081 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1141 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1201 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1261 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     1321 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1381 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     1441 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     1501 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     1561 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     1621 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     1681 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     1741 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     1801 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     1861 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     1921 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     1981 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2041 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2101 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2161 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2221 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     2281 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2341 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2401 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     2461 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     2521 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     2581 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     2641 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     2701 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     2761 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     2821 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag ggaagagcgg
     2881 accttctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc
     2941 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag
     3001 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc
     3061 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac
     3121 gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt
     3181 acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt
     3241 gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc
     3301 ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc
     3361 tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt
     3421 tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat
     3481 ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag
     3541 cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag
     3601 tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc
     3661 tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc
     3721 ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag
     3781 tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg
     3841 gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct
     3901 ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact
     3961 gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt
     4021 ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag
     4081 gtgtcgtgac gtacggccac catggaacgg gtgctcatgc accttagaaa gtccagagcc
     4141 gatctggaag ctgaggccag gggcgagggc gaaaccctgg ctaaacatag aaacatcctg
     4201 ctgaagctgg ccaaagaaag aaatctgaac atcgtgaaga tcagagagga agtgggcatc
     4261 agcggcgagt ctctgatcca cagacctgag atgctggaga cactgaagga ggtggaagag
     4321 ggactgtacg acgctgtgct gtgcatggac atcgacagac tgggcagagg caataccaag
     4381 gagcagggca ttatcctgga ggccttcaag aatagcaaca ccaagatcat cacccctctg
     4441 aaaacatacg acctgaacaa cgagttcgac gaggaatacg ccgagtttgg cgccttcatg
     4501 gccaggcggg aattcaagtt tatcaccaga agactgcagc ggggcagagt ggccacagtt
     4561 gaggagggaa attacatcgc tccaagacca ccttacggct acatcatcga gaaaaacaac
     4621 aaggagcggt acctgattcc tcaccctgag caggccccta tcgtgaagat gatctttgac
     4681 tggtacaccc acgacgatcc taacgtgcgg atgggcgcca gcaagatcgc caacgagctg
     4741 aacaagttct gcaagagccc tacaggcatc gcctggaagg gctccaccgt gttgtctatc
     4801 ctgaagaatg ccgtgtacgc cggcagaatc cagtggcgga agaaggagga aaaaaaatct
     4861 atcacaccag gcaaaaaaaa ggatgtccgg atgcggccca aggaacaatg gatcgacgtg
     4921 gagggcaagc acgaacctct ggtgtccatg gaaacctacc tgaaggccca agagatcctg
     4981 gagcggaagt accacgtgcc ctatcagatc cagaacggca tcactaaccc cctggccgga
     5041 ctggtcaagt gcggaatttg tggtagcagc atggtgtata gaccttacac ccaccagcgc
     5101 cacccccacc tgatctgcta caacagatac tgtaccaaca agtctagcag attcgagtac
     5161 gtggaaagca aaattatcca aggcctgcac cagctgctgg cacagtacaa ggccgattgg
     5221 ttcaaacatc agagacccaa ggtgaacgac gacagcgtgg acctgagaca gaaagccctc
     5281 caccggctgg agaaggaact gaatgacctg tacaagcaga aggacaacct gcacaatctg
     5341 ctggagaagg gcgtgtacag catcgataca ttcctggaaa gaagcaacat cctggctgaa
     5401 cgcatcgatg ataccaagaa ggccatccac gacgccgaga aggctctggc cgaggaggtg
     5461 cagcggaaca aagtgaagaa ggatatcatc cccacagtcg agaacgtgct tgaactgtac
     5521 tacaagaccc aggaccctgc caagaagaac aacctgctga agagcgtgct ggactacgct
     5581 gtttacagaa aagaaaagca ccagagagac gatgacttca ccttggtgct ttatcctaag
     5641 ctgcctcaga tcaacagc
//
