LOCUS       sequence-378308-        5673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Kp03 recombinase from Ef1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5673)
  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 5673)
  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: pAF0034 Ef1a-Kp03-2a-EGFP
FEATURES             Location/Qualifiers
     source          1..5673
                     /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"
     regulatory      3120..3129
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4713..4766
                     /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(4767..4769,4770..5483)
                     /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(4767..4769,4770..5480)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4809..4830)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     5417..5438
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5515..5636
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5552..5571)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5606..5625
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        join(5668..5673,1..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 ccaccatggg cagacaggtg atcacctacc tgcggttctc tagcaagccc caggagagag
     3181 gcgacagcat ccggagacaa aagggcctgt tcgagcggtg gctgaaggac aaccccgacg
     3241 ccaaggtggt ggacgagttc agcgacgagg gcgcttcagc ttatcacggc caccacctga
     3301 agggcgattt cggcagaatg ctgcagaaca tccaagacgg caagtacctg agcggccaga
     3361 cagtcctgct ggttgagtcc gaaaccagac tgaacagaca gaaggccaga aacaccgaga
     3421 acctggtcga ccttatcact ggaaaaggcg tggacgtgat ctgcctggaa agcggcaaaa
     3481 tctacaccag tacaaacatt gatgacctgg ataccagcat ccagctgaag atcgccgctc
     3541 acatcgccca ccagcagagc aaggaaaaga gcatcaaggt cagcgccgcc tgggagcaca
     3601 gagctcagct ggccctggaa ggcaagcagc agctgacaaa gaacgtgccc ggctggatcg
     3661 atcctgacac cagaaagctg aacgagcaca gcagcaccgt ggtgaccatt ttcgacctgc
     3721 tgctgagtgg cgagagcctg cacaacatcg ccagatacct gcaggccaac aacatcaagt
     3781 cttttagccg gcgggaaaag gccaatggct tcagcgtgca cagcgtgcgg accatcctga
     3841 gatctgagag caccgtgggc acccttgctg cctctaagag aaatgataga cctgccatcc
     3901 ccaactacta cgagcctgct atcgacgtgg ccaccttcaa caaggcccag gagatcctga
     3961 gcaaaaaccg ggtgggtaga gcccctgcca gcgacaatcc tatcacaatc aaccttttca
     4021 agggcatcat tagatgccag tgcggcgcca gcgtgcatcc taccggagtt aaggcaacct
     4081 accagggcgt gtacagatgt aacaacgtgc ccgatggcag atgcaacgtg cctacaatca
     4141 aaagaaagcc cttcgataag tggatgctgg acaacatagt tggatttctg gaacgggacg
     4201 acggcaacaa cacagacaag cggaaggctg aaatcgagta ccaaatcagc ctggtgacca
     4261 gcaaactgaa aaaggccaca acactgctgc tggaactgga cgacgtgacc gagctgaagg
     4321 aacaggtgaa ggagctgaac atccagcgga gcaacctgca atctgaactg gacgagctca
     4381 accagagaga gacactgagc gacaagcctc tgcaccacct gagcgagatc gacttgacca
     4441 ccaaggcagg cagagtggaa gcccagctga tcctgagtaa gttcgtgcag agtatcgagc
     4501 tccagagaga aatgatcatc atcaccctga gaaacggcac agtgatcggc aagtccagag
     4561 acctgagccc tgtgctgtct caggacctga tgaaacaggt cgtgtcctcc ccatctccaa
     4621 ccgacatcga catgttctca gtgataacaa gtgatgagga atttagaaaa agcggaaaac
     4681 aggtgaccaa gcggagcgct agcggcagtg gagagggcag aggaagtctg ctaacatgcg
     4741 gtgacgtcga ggagaatcct ggcccagtga gcaagggcga ggagctgttc accggggtgg
     4801 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     4861 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     4921 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     4981 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     5041 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     5101 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     5161 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     5221 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     5281 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     5341 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     5401 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     5461 gcatggacga gctgtacaag taagaattcg atatcaagct tatcgataat caacaacttg
     5521 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     5581 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttagacg
     5641 tcaggtggca cttttcgggg aaatgtgcgc gga
//
