LOCUS       sequence-355626-        5558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  attB-puro DNA donor for twinPE and Bxb1 mediated gene-size 
            insertion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5558)
  AUTHORS   Anzalone AV, Gao XD, Podracky CJ, Nelson AT, Koblan LW, Raguram A, 
            Levy JM, Mercer JAM, Liu DR
  TITLE     Programmable deletion, replacement, integration and inversion of 
            large DNA sequences with twin prime editing.
  JOURNAL   Nat Biotechnol. 2022 May;40(5):731-740. doi: 
            10.1038/s41587-021-01133-w. Epub 2021 Dec 9.
  PUBMED    34887556
REFERENCE   2  (bases 1 to 5558)
  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"; doi: 
            "10.1038/s41587-021-01133-w"; journalName: "Nat Biotechnol"; date: 
            "2022-05"; volume: "40"; issue: "5"; pages: "731-740"
COMMENT     Sequence Label: attB-puro donor.dna
FEATURES             Location/Qualifiers
     source          1..5558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    203..240
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             join(243..245,246..956)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(243..245,246..956)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(285..306)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(546..565)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     893..914
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      1002..1011
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        1118..1329
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1330..2268
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2225..2245
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2289..2885
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2289..2308)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2785..2805
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2895..2948
                     /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             2961..3659
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     polyA_signal    3673..3807
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3697..3716)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3751..3770
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3943..3990
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4009..4812
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPGGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4072..4091)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4682..4701
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4941..5529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5021..5040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gatgccagct cattcctccc actcatgatc tatagatccc ccgggctgca ggaattctac
       61 ccactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
      121 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
      181 ggcaagctta catcgagatc ccggcttgtc gacgacggcg gtctccgtcg tcaggatcat
      241 ccgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
      301 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
      361 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
      421 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
      481 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
      541 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
      601 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
      661 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
      721 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
      781 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
      841 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
      901 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagagcg
      961 gcctgaggag cagagcccag gcgagcaaca gcgccgtgga cgccaccatg ggcgatcgcc
     1021 cgggaattga ctagtgcggc cgcctaggat ccatgccgat agcgttggtt gagtggataa
     1081 ccgtattacc gccaagctta tgcatgtgcc cgtcagtggg cagagcgcac atcgcccaca
     1141 gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc
     1201 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     1261 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     1321 agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc
     1381 ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt
     1441 cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt
     1501 gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt
     1561 cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct
     1621 gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt
     1681 atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg
     1741 gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc
     1801 cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg
     1861 ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca
     1921 gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa
     1981 aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg
     2041 ccgtccaggc acctcgatta gttctcgcgc ttttggagta cgtcgtcttt aggttggggg
     2101 gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca
     2161 gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc
     2221 attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgagct
     2281 agcccaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc
     2341 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     2401 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     2461 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     2521 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     2581 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     2641 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     2701 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     2761 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     2821 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     2881 gtgccggatc gggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc
     2941 ctggcccacc ggtcgccacc agcgagctga ttaaggagaa catgcacatg aagctgtaca
     3001 tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct
     3061 acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct
     3121 tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg
     3181 gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca
     3241 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
     3301 tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga
     3361 agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg
     3421 aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacatca
     3481 agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg
     3541 tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg
     3601 aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattaag
     3661 aattctctag aggatccaga catgataaga tacattgatg agtttggaca aaccacaact
     3721 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     3781 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     3841 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg
     3901 gctgattatg atcctgcaag cctcgtcgcc gcggtttatt ctgttgacaa ttaatcatcg
     3961 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat gggatcggcc
     4021 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     4081 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     4141 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     4201 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4261 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4321 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     4381 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     4441 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4501 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccggcggc
     4561 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4621 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4681 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4741 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     4801 gagttcttct gagcgggact ctggggttcg aataaagacc gaccaagcga cgtctgagag
     4861 ctccctggcg aattcggtac caataaaaga gctttatttt catgatctgt gtgttggttt
     4921 ttggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4981 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5041 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5101 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5161 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5221 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5281 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5341 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5401 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5461 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5521 ggatctcaag aagatccttt gatcttttct agtgtgcg
//
