LOCUS       sequence-491687-        5708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of a human codon optimized engineered hifiDn29 
            recombinase (highest specificity 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-hifiDn29-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..5708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(97..116)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      910..1498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1399..1418
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1652..1669
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1714..2892
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1945..2883
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2840..2860
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4494..4511
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4527..4580
                     /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(4581..4583,4584..5297)
                     /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(4581..4583,4584..5294)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4623..4644)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4884..4903)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5231..5252
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5329..5450
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5366..5385)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5420..5439
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        5482..5586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5587..5655,5656..5708,1..739)
                     /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 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
       61 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      121 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      181 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      241 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
      301 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
      361 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
      421 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
      481 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
      541 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
      601 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
      661 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
      721 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
      781 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
      841 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
      901 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
      961 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1021 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     1081 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1141 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1201 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1261 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1321 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1381 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     1441 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     1501 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     1561 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     1621 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagggaagag
     1681 cggaccttct aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag
     1741 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc
     1801 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
     1861 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
     1921 aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc
     1981 tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt
     2041 cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga
     2101 gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga
     2161 atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat
     2221 ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa
     2281 gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc
     2341 cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg
     2401 tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg
     2461 ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt
     2521 cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt
     2581 gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc
     2641 acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg
     2701 tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag
     2761 actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag
     2821 tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt
     2881 caggtgtcgt gacgtacggc caccgtttaa ctttaagaag gagatatacc atggaacggg
     2941 tgctcataca ccttagaaag tccagagccg atctggaagc tgaggccagg ggcgagggcg
     3001 aaaccctggc taaacataga aacatcctgc tgaagctggc caaagaaaga aatctgaaca
     3061 tcgtgaagat cagagaggaa gtgggcatca gcggcgagtc tctgatccac agacctgaga
     3121 tgctggagac actgaagggg gtggaagagg gactgtacga cgctgtgctg tgcatggaca
     3181 tcgacagact gggcagaggc aataccaagg agcagggcat tgtgctggag gccttcaaga
     3241 atagcaacac caagatcatc acccctctga aaacatacga cctgaacaac gagttcgacg
     3301 aggaatacgc cgagtttggc gccttcatgg ccaggcggga attcaagttt atcaccagaa
     3361 gactgcagcg gggcagagtg gccacagttg aggagggaaa ttacatcgct ccaagaccac
     3421 cttacggcta catcatcgag aaaaacaaca aggagcggta cctgattcct caccctgagc
     3481 aggcccctat cgtgaagatg atctttgact ggtacaccca cgacgatcct aacgtgcgga
     3541 tgggcgccag caagatcgcc aacgagctga acaagttctg caagagccct acaggcatcc
     3601 cctggaaggt ctccaccgtg ttgtctaagt tgaagaatgc cgtgtacgcc ggcagaatcc
     3661 agtggcggaa gcgggaggaa aaaaaatcta tcacaccagg caaaaaaaag gatgtccgga
     3721 tgcggcccaa ggaacaatgg atcgacgtgg agggcaagca cgaacctctg gtgtccatgg
     3781 aaacctacct gaaggcccaa gagatcctgg agcggaagta ccacgtgccc tatcagaagc
     3841 agaacggcat cactaacccc ctggccggac tggtcaagtg cgggatttgt ggtagcagca
     3901 tggtgtatag accttacacc cacaagcgcc acccccacct gatctgctac aaaagatact
     3961 gtaccaacaa gtctagcaga ttcgagtacg tggaaagcaa aattatccaa ggcctgcacc
     4021 agctgctggc acagtacaag gccgattggt tcaaacatca gagacccaag gtgaacgacg
     4081 acagcgtgga cctgagacag aaagcccccc accggctgga gaaggaactg aatgacctgt
     4141 acaagcagaa ggacaacctg cacaatctgc tggagaaggg cgtgtacagc atcgatacat
     4201 tcctggaaag aagcaacatc ctggctgaac gcatcgatga taccaagaag gccatccacg
     4261 acgccgagaa ggctctggcc gaggaggtgc agcggaacaa agtgaagaag gatatcatcc
     4321 ccacagtcga gaacgtgctt gaactgtact acaagaccca ggaccctgcc aagaagaaca
     4381 acctgctgaa gagcgtgctg gactacgctg tttacagaaa agaaaagcac cagagaaacg
     4441 atgacttcac cttggtgctt tatcctaagc tgcctcagat caacagcggc ggtcaccatc
     4501 accaccacca cgctagcggc agtggagagg gcagaggaag tctgctaaca tgcggtgacg
     4561 tcgaggagaa tcctggccca gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     4621 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     4681 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     4741 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     4801 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     4861 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     4921 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     4981 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     5041 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     5101 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     5161 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     5221 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     5281 acgagctgta caagtaagaa ttcgatatca agcttatcga taatcaacaa cttgtttatt
     5341 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     5401 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta gacgtcaggt
     5461 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5521 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5581 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5641 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5701 ggtgcacg
//
