LOCUS       sequence-491685-        5708 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  Mammalian expression of a human codon optimized engineered superDn29
            recombinase (highest efficiency 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, Durrant MG, Wei 
            J, Pawluk A, Konermann S, Marson A, Gilbert LA, Hsu PD
  TITLE     Site-specific DNA insertion into the human genome with engineered 
            recombinases
  JOURNAL   bioRxiv 2024.11.01.621560
REFERENCE   2  (bases 1 to 5708)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.11.01.621560"
COMMENT     Sequence Label: pEf1a-superDn29-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..5708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     141..160
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(379..483)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    515..636
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(526..545)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     580..599
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(668..1381,1382..1384))
                     /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(671..1381,1382..1384))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(713..734)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1062..1081
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1321..1342
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1385..1438)
                     /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(1454..1471)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(3073..4251)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3082..4020
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3105..3125)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(4296..4313)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4467..5055)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4547..4566)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5226..5708,1..309,310..378))
                     /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 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
       61 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
      121 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
      181 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
      241 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
      301 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
      361 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
      421 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      481 gcgcacattt ccccgaaaag tgccacctga cgtctaagat acattgatga gtttggacaa
      541 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
      601 ttatttgtaa ccattataag ctgcaataaa caagttgttg attatcgata agcttgatat
      661 cgaattctta cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact
      721 ccagcaggac catgtgatcg cgcttctcgt tggggtcttt gctcagggcg gactgggtgc
      781 tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt
      841 agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg aagttcacct
      901 tgatgccgtt cttctgcttg tcggccatga tatagacgtt gtggctgttg tagttgtact
      961 ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc
     1021 ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt
     1081 ccttgaagaa gatggtgcgc tcctggacgt agccttcggg catggcggac ttgaagaagt
     1141 cgtgctgctt catgtggtcg gggtagcggc tgaagcactg cacgccgtag gtcagggtgg
     1201 tcacgagggt gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca
     1261 gcttgccgta ggtggcatcg ccctcgccct cgccggacac gctgaacttg tggccgttta
     1321 cgtcgccgtc cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc
     1381 tcactgggcc aggattctcc tcgacgtcac cgcatgttag cagacttcct ctgccctctc
     1441 cactgccgct agcgtggtgg tggtgatggt gaccgccgct gttgatctga ggcagcttag
     1501 gataaagcac caaggtgaag tcatcgtctc tctggtgctt ttcttttctg taaacagcgt
     1561 agtccagcac gctcttcagc aggttgttct tcttggcagg gtcctgggtc ttgtagtaca
     1621 gttcaagcac gttctcgact gtggggatga tatccttctt cactttgttc cgctgcacct
     1681 cctcggccag agccttctcg gcgtcgtgga tggccttctt ggtatcatcg atgcgttcag
     1741 ccaggatgtt gcttctttcc aggaatgtat cgatgctgta cacgcccttc tccagcagat
     1801 tgtgcaggtt gtccttctgc ttgcacaggt cattcagttc cttctccagc cggtggaggg
     1861 ctttctgtct ggggtccacg ctgtcgtcgt tcaccttggg tctctgatgt ttgaaccaat
     1921 cggccttgta ctgtgccagc agctggtgca ggccttggat aattttgctt tccacgtact
     1981 cgaatctgct agacttgttg gtacagtatc tctggtagca gatcaggtgg gggtggcgct
     2041 tgtgggtgta aggtctatac accatgctgc taccacaaat cccgcacttg accagtccgg
     2101 ccagggggtt agtgatgccg ttctgcttct gatagggcac gtggtacttc cgctccagga
     2161 tctcttgggc cttcaggtag gtttccatgg acaccagagg ttcgtgcttg ccctccacgt
     2221 cgatccattg ttccttgggc cgcatccgga catccttttt tttgcctggt gtgatagatt
     2281 ttttttcctc ccgcttccgc cactggattc tgccggcgta cacggcattc ttcaacttag
     2341 acaacacggt ggagaccttc caggggatgc ctgtagggct cttgcagaac ttgttcagct
     2401 cgttggcgat cttgctggcg cccatccgca cgttaggatc gtcgtgggtg taccagtcaa
     2461 agatcatctt cacgataggg gcctgctcag ggtgaggaat caggtaccgc tccttgttgt
     2521 ttttctcgat gatgtagccg taaggtggtc ttggagcgat gtaatttccc tcctcaactg
     2581 tggccactct gccccgctgc agtcttctgg tgataaactt gaattcccgc ctggccatga
     2641 aggcgccaaa ctcggcgtat tcctcgtcga actcgttgtt caggtcgtat gttttcagag
     2701 gggtgatgat cttggtgttg ctattcttga aggcctccag gataatgccc tgctccttgg
     2761 tattgcctct gcccagtctg tcgatgtcca tgcacagcac agcgtcgtac agtccctctt
     2821 ccaccccctt cagtgtctcc agcatctcag gtctgtggat cagagactcg ccgctgatgc
     2881 ccacttcctc tctgatcttc acgatgttca gatttctttc tttggccagc ttcagcagga
     2941 tgtttctatg tttagccagg gtttcgccct cgcccctggc ctcagcttcc agatcggctc
     3001 tggactttct aaggtgtatg agcacccgtt ccatggtata tctccttctt aaagttaaac
     3061 ggtggccgta cgtcacgaca cctgaaatgg aagaaaaaaa ctttgaacca ctgtctgagg
     3121 cttgagaatg aaccaagatc caaactcaaa aagggcaaat tccaaggaga attacatcaa
     3181 gtgccaagct ggcctaactt cagtctccac ccactcagtg tggggaaact ccatcgcata
     3241 aaacccctcc ccccaaccta aagacgacgt actccaaaag ctcgagaact aatcgaggtg
     3301 cctggacggc gcccggtact ccgtggagtc acatgaagcg acggctgagg acggaaaggc
     3361 ccttttcctt tgtgtgggtg actcacccgc ccgctctccc gagcgccgcg tcctccattt
     3421 tgagctccct gcagcagggc cgggaagcgg ccatctttcc gctcacgcaa ctggtgccga
     3481 ccgggccagc cttgccgccc agggcggggc gatacacggc ggcgcgaggc caggcaccag
     3541 agcaggccgg ccagcttgag actacccccg tccgattctc ggtggccgcg ctcgcaggcc
     3601 ccgcctcgcc gaacatgtgc gctgggacgc acgggccccg tcgccgcccg cggccccaaa
     3661 aaccgaaata ccagtgtgca gatcttggcc cgcatttaca agactatctt gccagaaaaa
     3721 aagcgtcgca gcaggtcatc aaaaatttta aatggctaga gacttatcga aagcagcgag
     3781 acaggcgcga aggtgccacc agattcgcac gcggcggccc cagcgcccag gccaggcctc
     3841 aactcaagca cgaggcgaag gggctcctta agcgcaaggc ctcgaactct cccacccact
     3901 tccaacccga agctcgggat caagaatcac gtactgcagc caggtggaag taattcaagg
     3961 cacgcaaggg ccataacccg taaagaggcc aggcccgcgg gaaccacaca cggcacttac
     4021 ctgtgttctg gcggcaaacc cgttgcgaaa aagaacgttc acggcgacta ctgcacttat
     4081 atacggttct cccccaccct cgggaaaaag gcggagccag tacacgacat cactttccca
     4141 gtttaccccg cgccaccttc tctaggcacc ggttcaattg ccgacccctc cccccaactt
     4201 ctcggggact gtgggcgatg tgcgctctgc ccactgacgg gcaccggagc caattcccac
     4261 tcctttcaag acctagaagg tccgctcttc ccttcctcgc tcactgactc gctgcgctcg
     4321 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4381 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4441 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4501 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4561 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4621 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4681 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4741 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4801 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4861 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4921 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4981 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5041 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5101 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5161 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5221 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5281 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5341 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5401 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5461 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5521 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5581 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5641 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5701 tcactcat
//
