LOCUS       sequence-378312-        5646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Nm60 recombinase from Ef1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5646)
  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 5646)
  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: pAF0035 Ef1a-Nm60-2a-EGFP
FEATURES             Location/Qualifiers
     source          1..5646
                     /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             4686..4739
                     /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(4740..4742,4743..5456)
                     /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(4740..4742,4743..5453)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4782..4803)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5043..5062)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5390..5411
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5488..5609
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5525..5544)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5579..5598
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        join(5641..5646,1..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 ccaccatgag cagacctaca ggcctgacca ttgacatcta cctgcggaaa tctagaaagg
     3181 atctggaaga agagaagaaa gccagcgaga gcggcgaaac ctacgataca ctggaaagac
     3241 acagacggac cctcttcgcc gtggctaaga aagagcggca caacatcgct aacatctatg
     3301 aggaagtcgt gtccggcgag tccgtgtctg agcgaccaca gatccaagct atgctgagaa
     3361 acctggaaac cagccacatc gagggcgtgc tggttatgga cctggataga ctgggcagag
     3421 gcgacatgct ggaccaggga ctgctggaca gagccttcag atacagcgga gccaagatcc
     3481 tgacccctac cgaggtgtac gaccccgaga gcgagacatg ggagctggtg ttcggcatca
     3541 agtctctggt ggccagagag gaactgaaag ccatcacaag aaggatgcag agaggaagag
     3601 tggcctctgc cggcgaggga aagtccatca gcaaggtgcc accttacggc tatctgcgcg
     3661 atgagaacct gagactgtac cctgatcctg aaaccgcctg ggtggtgaag aaaatgttcg
     3721 agatgatgag agatggccac ggcaggatcg ccgtggccca ggagctggac aagctgggaa
     3781 tcaaacctcc taatgataag cggcggagct gggcccctag cagcatcacc gccatcatca
     3841 agaacgaggt ctacctgggc accatcattt ggggccaggt gaagtacagc aagcggaacg
     3901 gcaagtacaa gaagaccaag ctgcctagat ctaagtggac catcaaggaa aacgctcatg
     3961 agcctatcgt gtccagagaa ctcttcgagg ccgctaatcg ggcccacaca ggcagatggc
     4021 ggccttcaac aaacgccaca aagaccctga gcaaccccct ggctggcgtt ctgaagtgcg
     4081 acgtgtgcgg cttcaccatg ctgtaccagc ccagacctaa ccggcccaat gacttcatcc
     4141 ggtgcaccca gcctacctgt caggccgtgc agaagggcgc caccctggcc ctggtggaac
     4201 agcacatcct ggacggcctg aagcaatttg cccaggagct ggaactgcaa acagaagtgc
     4261 ccgagctgga caacgacaag gacatcgcag tgaaaaagag cctggtgggg aacaagcagg
     4321 aggaaatcgc ccagctggaa acacagaaaa gcaagctgca cgacctgctt gaacgcggca
     4381 tctacgacgt ggacaccttc ctggaaagac agcagaacct gaataaccgg atcaacggcc
     4441 tgcaggacga catcagaaac atcgagtctg agatcaagaa agaagaggtg cggaacagca
     4501 gcgtgctgaa tctgctgccg caacttcaga ccgtgatcag cgagtacgag aacgccgata
     4561 ccgaaagcaa gaacagactg ctgaagagcg tgctggaaaa ggccacatac ctgagaaaga
     4621 aagagtggac aaagagagat cagtttatca ttcagctgta ccccaagatc gctagcggca
     4681 gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccag
     4741 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     4801 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     4861 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     4921 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     4981 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     5041 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     5101 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     5161 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     5221 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     5281 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     5341 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     5401 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaagaat
     5461 tcgatatcaa gcttatcgat aatcaacaac ttgtttattg cagcttataa tggttacaaa
     5521 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     5581 ggtttgtcca aactcatcaa tgtatcttag acgtcaggtg gcacttttcg gggaaatgtg
     5641 cgcgga
//
