LOCUS       sequence-491691-        5708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of a human codon optimized engineered goldDn29 
            recombinase (efficient and specific variant).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5708)
  AUTHORS   Fanton A, Bartie LJ, Martins JQ, Tran VQ, Goudy L, Kernick C, 
            Durrant MG, Wei J, Armour-Garb Z, Pawluk A, Konermann S, Marson A, 
            Gilbert LA, Roth TL, Hsu PD
  TITLE     Site-specific DNA insertion into the human genome with engineered 
            recombinases.
  JOURNAL   Nat Biotechnol. 2025 Nov 6. doi: 10.1038/s41587-025-02895-3.
  PUBMED    41199024
REFERENCE   2  (bases 1 to 5708)
  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. 2025 Nov 6. doi: 10.1038/s41587-025-02895-3."
COMMENT     Sequence Label: pEf1a-goldDn29-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..5708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(80..871,872..940))
                     /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     703..722
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(941..1045)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    1077..1198
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1088..1107)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1142..1161
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(1230..1943,1944..1946))
                     /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             complement(join(1233..1943,1944..1946))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1275..1296)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1624..1643
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1883..1904
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1947..2000)
                     /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             complement(2016..2033)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(3635..4813)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3644..4582
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3667..3687)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(4858..4875)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5029..5617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5109..5128)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
       61 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
      121 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
      181 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
      241 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
      301 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
      361 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
      421 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
      481 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
      541 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
      601 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
      661 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
      721 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
      781 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
      841 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
      901 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
      961 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     1021 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     1081 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     1141 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgt
     1201 tgattatcga taagcttgat atcgaattct tacttgtaca gctcgtccat gccgagagtg
     1261 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct
     1321 ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg
     1381 ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg
     1441 tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg
     1501 ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag
     1561 tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg
     1621 cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg
     1681 ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac
     1741 tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg
     1801 gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac
     1861 acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg
     1921 gtgaacagct cctcgccctt gctcactggg ccaggattct cctcgacgtc accgcatgtt
     1981 agcagacttc ctctgccctc tccactgccg ctagcgtggt ggtggtgatg gtgaccgccg
     2041 ctgttgatct gaggcagctt aggataaagc accaaggtga agtcatcgtt tctctggtgc
     2101 ttttcttttc tgtaaacagc gtagtccagc acgctcttca gcaggttgtt cttcttggca
     2161 gggtcctggg tcttgtagta cagttcaagc acgttctcga ctgtggggat gatatccttc
     2221 ttcactttgt tccgctgcac ctcctcggcc agagccttct cggcgtcgtg gatggccttc
     2281 ttggtatcat cgatgcgttc agccaggatg ttgcttcttt ccaggaatgt atcgatgctg
     2341 tacacgccct tctccagcag attgtgcagg ttgtccttct gcttgtacag gtcattcagt
     2401 tccttctcca gccggtgggg ggctttctgt ctcaggtcca cgctgtcgtc gttcaccttg
     2461 ggtctctgat gtttgaacca atcggccttg tactgtgcca gcagctggtg caggccttgg
     2521 ataattttgc tttccacgta ctcgaatctg ctagacttgt tggtacagta tcttttgtag
     2581 cagatcaggt gggggtggcg cttgtgggtg taaggtctat acaccatgct gctaccacaa
     2641 atcccgcact tgaccagtcc ggccaggggg ttagtgatgc cgttctggat ctgatagggc
     2701 acgtggtact tccgctccag gatctcttgg gccttcaggt aggtttccat ggacaccaga
     2761 ggttcgtgct tgccctccac gtcgatccat tgttccttgg gccgcatccg gacatccttt
     2821 tttttgcctg gtgtgataga ttttttttcc tccttcttcc gccactggat tctgccggcg
     2881 tacacggcat tcttcaagat agacaacacg gtggagacct tccaggggat gcctgtaggg
     2941 ctcttgcaga acttgttcag ctcgttggcg atcttgctgg cgcccatccg cacgttagga
     3001 tcgtcgtggg tgtaccagtc aaagatcatc ttcacgatag gggcctgctc agggtgagga
     3061 atcaggtacc gctccttgtt gtttttctcg atgatgtagc cgtaaggtgg tcttggagcg
     3121 atgtaatttc cctcctcaac tgtggccact ctgccccgct gcagtcttct ggtgataaac
     3181 ttgaattccc gcctggccat gaaggcgcca aactcggcgt attcctcgtc gaactcgttg
     3241 ttcaggtcgt atgttttcag aggggtgatg atcttggtgt tgctattctt gaaggcctcc
     3301 aggataatgc cctgctcctt ggtattgcct ctgcccagtc tgtcgatgtc catgcacagc
     3361 acagcgtcgt acagtccctc ttccaccccc ttcagtgtct ccagcatctc aggtctgtgg
     3421 atcagagact cgccgctgat gcccacttcc tctctgatct tcacgatgtt cagatttctt
     3481 tctttggcca gcttcagcag gatgtttcta tgtttagcca gggtttcgcc ctcgcccctg
     3541 gcctcagctt ccagatcggc tctggacttt ctaaggtgta tgagcacccg ttccatggta
     3601 tatctccttc ttaaagttaa acggtggccg tacgtcacga cacctgaaat ggaagaaaaa
     3661 aactttgaac cactgtctga ggcttgagaa tgaaccaaga tccaaactca aaaagggcaa
     3721 attccaagga gaattacatc aagtgccaag ctggcctaac ttcagtctcc acccactcag
     3781 tgtggggaaa ctccatcgca taaaacccct ccccccaacc taaagacgac gtactccaaa
     3841 agctcgagaa ctaatcgagg tgcctggacg gcgcccggta ctccgtggag tcacatgaag
     3901 cgacggctga ggacggaaag gcccttttcc tttgtgtggg tgactcaccc gcccgctctc
     3961 ccgagcgccg cgtcctccat tttgagctcc ctgcagcagg gccgggaagc ggccatcttt
     4021 ccgctcacgc aactggtgcc gaccgggcca gccttgccgc ccagggcggg gcgatacacg
     4081 gcggcgcgag gccaggcacc agagcaggcc ggccagcttg agactacccc cgtccgattc
     4141 tcggtggccg cgctcgcagg ccccgcctcg ccgaacatgt gcgctgggac gcacgggccc
     4201 cgtcgccgcc cgcggcccca aaaaccgaaa taccagtgtg cagatcttgg cccgcattta
     4261 caagactatc ttgccagaaa aaaagcgtcg cagcaggtca tcaaaaattt taaatggcta
     4321 gagacttatc gaaagcagcg agacaggcgc gaaggtgcca ccagattcgc acgcggcggc
     4381 cccagcgccc aggccaggcc tcaactcaag cacgaggcga aggggctcct taagcgcaag
     4441 gcctcgaact ctcccaccca cttccaaccc gaagctcggg atcaagaatc acgtactgca
     4501 gccaggtgga agtaattcaa ggcacgcaag ggccataacc cgtaaagagg ccaggcccgc
     4561 gggaaccaca cacggcactt acctgtgttc tggcggcaaa cccgttgcga aaaagaacgt
     4621 tcacggcgac tactgcactt atatacggtt ctcccccacc ctcgggaaaa aggcggagcc
     4681 agtacacgac atcactttcc cagtttaccc cgcgccacct tctctaggca ccggttcaat
     4741 tgccgacccc tccccccaac ttctcgggga ctgtgggcga tgtgcgctct gcccactgac
     4801 gggcaccgga gccaattccc actcctttca agacctagaa ggtccgctct tcccttcctc
     4861 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4921 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4981 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5041 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5101 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5161 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5221 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5281 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5341 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5401 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5461 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5521 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5581 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5641 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5701 aaaaagga
//
