LOCUS       sequence-492676-        5188 bp    DNA     circular SYN 12-NOV-2025
DEFINITION  In vitro transcription of human codon optimized engineered 
            hifiDn29-P2A-GFP .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5188)
  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 5188)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 12, 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: pIVT_hifiDn29-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..5188
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(join(1318..2031,2032..2034))
                     /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(1321..2031,2032..2034))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1363..1384)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1712..1731
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1971..1992
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2035..2088)
                     /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(2104..2121)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     3955..3974
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4053..4157
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4158..4226,4227..5018)
                     /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(4376..4395)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccctag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtatc gagccccagc tggttctttc cgcctcagaa ggaagacgaa ttcgaagact
     1081 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
     1141 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
     1201 tttttttttt tttttttttt ttttcttcct actcaggctt tattcaaaga ccaagaggta
     1261 caggtgcaag ggagagaaga agggcatggc cagaaggcaa gccccgcaga aggcagctta
     1321 cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac
     1381 catgtgatcg cgcttctcgt tggggtcttt gctcagggcg gactgggtgc tcaggtagtg
     1441 gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc
     1501 gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg aagttcacct tgatgccgtt
     1561 cttctgcttg tcggccatga tatagacgtt gtggctgttg tagttgtact ccagcttgtg
     1621 ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag
     1681 ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa
     1741 gatggtgcgc tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt
     1801 catgtggtcg gggtagcggc tgaagcactg cacgccgtag gtcagggtgg tcacgagggt
     1861 gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta
     1921 ggtggcatcg ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc
     1981 cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcactgggcc
     2041 aggattctcc tcgacgtcac cgcatgttag cagacttcct ctgccctctc cactgccgct
     2101 agcgtggtgg tggtgatggt gaccgccgct gttgatctga ggcagcttag gataaagcac
     2161 caaggtgaag tcatcgtttc tctggtgctt ttcttttctg taaacagcgt agtccagcac
     2221 gctcttcagc aggttgttct tcttggcagg gtcctgggtc ttgtagtaca gttcaagcac
     2281 gttctcgact gtggggatga tatccttctt cactttgttc cgctgcacct cctcggccag
     2341 agccttctcg gcgtcgtgga tggccttctt ggtatcatcg atgcgttcag ccaggatgtt
     2401 gcttctttcc aggaatgtat cgatgctgta cacgcccttc tccagcagat tgtgcaggtt
     2461 gtccttctgc ttgtacaggt cattcagttc cttctccagc cggtgggggg ctttctgtct
     2521 caggtccacg ctgtcgtcgt tcaccttggg tctctgatgt ttgaaccaat cggccttgta
     2581 ctgtgccagc agctggtgca ggccttggat aattttgctt tccacgtact cgaatctgct
     2641 agacttgttg gtacagtatc ttttgtagca gatcaggtgg gggtggcgct tgtgggtgta
     2701 aggtctatac accatgctgc taccacaaat cccgcacttg accagtccgg ccagggggtt
     2761 agtgatgccg ttctgcttct gatagggcac gtggtacttc cgctccagga tctcttgggc
     2821 cttcaggtag gtttccatgg acaccagagg ttcgtgcttg ccctccacgt cgatccattg
     2881 ttccttgggc cgcatccgga catccttttt tttgcctggt gtgatagatt ttttttcctc
     2941 ccgcttccgc cactggattc tgccggcgta cacggcattc ttcaacttag acaacacggt
     3001 ggagaccttc caggggatgc ctgtagggct cttgcagaac ttgttcagct cgttggcgat
     3061 cttgctggcg cccatccgca cgttaggatc gtcgtgggtg taccagtcaa agatcatctt
     3121 cacgataggg gcctgctcag ggtgaggaat caggtaccgc tccttgttgt ttttctcgat
     3181 gatgtagccg taaggtggtc ttggagcgat gtaatttccc tcctcaactg tggccactct
     3241 gccccgctgc agtcttctgg tgataaactt gaattcccgc ctggccatga aggcgccaaa
     3301 ctcggcgtat tcctcgtcga actcgttgtt caggtcgtat gttttcagag gggtgatgat
     3361 cttggtgttg ctattcttga aggcctccag cacaatgccc tgctccttgg tattgcctct
     3421 gcccagtctg tcgatgtcca tgcacagcac agcgtcgtac agtccctctt ccaccccctt
     3481 cagtgtctcc agcatctcag gtctgtggat cagagactcg ccgctgatgc ccacttcctc
     3541 tctgatcttc acgatgttca gatttctttc tttggccagc ttcagcagga tgtttctatg
     3601 tttagccagg gtttcgccct cgcccctggc ctcagcttcc agatcggctc tggactttct
     3661 aaggtgtatg agcacccgtt ccattcttat atttcttctt actcttcttt tctctcttat
     3721 ttccttatag tgtgtcgtat tagcggccgc ggatctctag cggatctgac ggttcactaa
     3781 accaacgcgt atatctggcc cgtacatcgc gaagcagcgc aaaacgccta accctaagca
     3841 gattcttcat gcaattgtcg gtcaagcctt gccttgttgt agcttaaatt ttgctcgcgc
     3901 actactcagc gacctccaac acacaagcag ggagcagata ctggcttaac tatgcggcat
     3961 cagagcagat tgtactgaga gtcgacccta ggggatcggg agatcgatct cccgatccgt
     4021 cgacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4081 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4141 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4201 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4261 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4321 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4381 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4441 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4501 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4561 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4621 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4681 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4741 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4801 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4861 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4921 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4981 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5041 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5101 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5161 accccgtaga aaagatcaaa ggatcttc
//
