LOCUS       sequence-491684-        5708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of a human codon optimized wildtype Dn29 
            recombinase.
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-Dn29-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..5708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(113..132)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      926..1514
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1415..1434
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1668..1685
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1730..2908
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1961..2899
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2856..2876
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4510..4527
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4543..4596
                     /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(4597..4599,4600..5313)
                     /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(4597..4599,4600..5310)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4639..4660)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4900..4919)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5247..5268
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5345..5466
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5382..5401)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5436..5455
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        5498..5602
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5603..5671,5672..5708,1..755)
                     /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"
ORIGIN
        1 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
       61 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
      121 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
      181 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
      241 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
      301 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
      361 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
      421 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
      481 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
      541 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
      601 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
      661 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
      721 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
      781 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
      841 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
      901 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      961 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     1021 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     1081 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     1141 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     1201 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     1261 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     1321 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     1381 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     1441 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     1501 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     1561 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     1621 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     1681 gcgaggaagg gaagagcgga ccttctaggt cttgaaagga gtgggaattg gctccggtgc
     1741 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     1801 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     1861 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     1921 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc
     1981 ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg
     2041 gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag
     2101 gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg
     2161 gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc
     2221 tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct
     2281 tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga
     2341 cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc
     2401 gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc
     2461 gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc
     2521 ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc
     2581 gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt
     2641 cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag
     2701 cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca
     2761 cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga
     2821 atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag
     2881 tttttttctt ccatttcagg tgtcgtgacg tacggccacc gtttaacttt aagaaggaga
     2941 tataccatgg aacgggtgct catgcacctt agaaagtcca gagccgatct ggaagctgag
     3001 gccaggggcg agggcgaaac cctggctaaa catagaaaca tcctgctgaa gctggccaaa
     3061 gaaagaaatc tgaacatcgt gaagatcaga gaggaagtgg gcatcagcgg cgagtctctg
     3121 atccacagac ctgagatgct ggagacactg aaggaggtgg aagagggact gtacgacgct
     3181 gtgctgtgca tggacatcga cagactgggc agaggcaata ccaaggagca gggcattatc
     3241 ctggaggcct tcaagaatag caacaccaag atcatcaccc ctctgaaaac atacgacctg
     3301 aacaacgagt tcgacgagga atacgccgag tttggcgcct tcatggccag gcgggaattc
     3361 aagtttatca ccagaagact gcagcggggc agagtggcca cagttgagga gggaaattac
     3421 atcgctccaa gaccacctta cggctacatc atcgagaaaa acaacaagga gcggtacctg
     3481 attcctcacc ctgagcaggc ccctatcgtg aagatgatct ttgactggta cacccacgac
     3541 gatcctaacg tgcggatggg cgccagcaag atcgccaacg agctgaacaa gttctgcaag
     3601 agccctacag gcatcgcctg gaagggctcc accgtgttgt ctatcctgaa gaatgccgtg
     3661 tacgccggca gaatccagtg gcggaagaag gaggaaaaaa aatctatcac accaggcaaa
     3721 aaaaaggatg tccggatgcg gcccaaggaa caatggatcg acgtggaggg caagcacgaa
     3781 cctctggtgt ccatggaaac ctacctgaag gcccaagaga tcctggagcg gaagtaccac
     3841 gtgccctatc agatccagaa cggcatcact aaccccctgg ccggactggt caagtgcgga
     3901 atttgtggta gcagcatggt gtatagacct tacacccacc agcgccaccc ccacctgatc
     3961 tgctacaaca gatactgtac caacaagtct agcagattcg agtacgtgga aagcaaaatt
     4021 atccaaggcc tgcaccagct gctggcacag tacaaggccg attggttcaa acatcagaga
     4081 cccaaggtga acgacgacag cgtggacctg agacagaaag ccctccaccg gctggagaag
     4141 gaactgaatg acctgtacaa gcagaaggac aacctgcaca atctgctgga gaagggcgtg
     4201 tacagcatcg atacattcct ggaaagaagc aacatcctgg ctgaacgcat cgatgatacc
     4261 aagaaggcca tccacgacgc cgagaaggct ctggccgagg aggtgcagcg gaacaaagtg
     4321 aagaaggata tcatccccac agtcgagaac gtgcttgaac tgtactacaa gacccaggac
     4381 cctgccaaga agaacaacct gctgaagagc gtgctggact acgctgttta cagaaaagaa
     4441 aagcaccaga gagacgatga cttcaccttg gtgctttatc ctaagctgcc tcagatcaac
     4501 agcggcggtc accatcacca ccaccacgct agcggcagtg gagagggcag aggaagtctg
     4561 ctaacatgcg gtgacgtcga ggagaatcct ggcccagtga gcaagggcga ggagctgttc
     4621 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     4681 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     4741 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     4801 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     4861 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     4921 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     4981 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     5041 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     5101 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     5161 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     5221 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     5281 atcactctcg gcatggacga gctgtacaag taagaattcg atatcaagct tatcgataat
     5341 caacaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5401 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5461 atcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5521 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5581 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5641 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5701 tgctgaag
//
