LOCUS       sequence-355630-        5568 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  attP-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 5568)
  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 5568)
  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: attP-puro donor
FEATURES             Location/Qualifiers
     source          1..5568
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    203..250
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             join(253..255,256..966)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(253..255,256..966)
                     /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(295..316)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(556..575)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     903..924
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      1012..1021
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        1128..1339
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1340..2278
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2235..2255
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2299..2895
                     /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(2299..2318)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2795..2815
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2905..2958
                     /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             2971..3669
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     polyA_signal    3683..3817
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3707..3726)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3761..3780
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3953..4000
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4019..4822
                     /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(4082..4101)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4692..4711
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4951..5539)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5031..5050)
                     /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 ccggtttgtc tggtcaacca ccgcggtctc agtggtgtac
      241 ggtacaaacc ccgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
      301 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
      361 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
      421 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
      481 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
      541 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
      601 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
      661 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
      721 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
      781 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
      841 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
      901 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
      961 tacaagagcg gcctgaggag cagagcccag gcgagcaaca gcgccgtgga cgccaccatg
     1021 ggcgatcgcc cgggaattga ctagtgcggc cgcctaggat ccatgccgat agcgttggtt
     1081 gagtggataa ccgtattacc gccaagctta tgcatgtgcc cgtcagtggg cagagcgcac
     1141 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag
     1201 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     1261 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     1321 gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac
     1381 gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga
     1441 tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc
     1501 ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg
     1561 gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg
     1621 atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct
     1681 gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg
     1741 cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc
     1801 tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg
     1861 ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg
     1921 ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc
     1981 acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga
     2041 gtaccgggcg ccgtccaggc acctcgatta gttctcgcgc ttttggagta cgtcgtcttt
     2101 aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga
     2161 agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg
     2221 atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt
     2281 gtcgtgagct agcccaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     2341 gacgtccccc gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     2401 cacaccgtcg acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     2461 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     2521 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     2581 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     2641 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     2701 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     2761 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     2821 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     2881 cgcaagcccg gtgccggatc gggagagggc agaggaagtc tgctaacatg cggtgacgtc
     2941 gaggagaatc ctggcccacc ggtcgccacc agcgagctga ttaaggagaa catgcacatg
     3001 aagctgtaca tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa
     3061 ggcaagccct acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc
     3121 cccttcgcct tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac
     3181 cacacccagg gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag
     3241 agagtcacca catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag
     3301 gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct
     3361 gtgatgcaga agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac
     3421 ggcggcctgg aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc
     3481 gcaaacatca agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc
     3541 gtctactatg tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc
     3601 gagcagcacg aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaag
     3661 cttaattaag aattctctag aggatccaga catgataaga tacattgatg agtttggaca
     3721 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     3781 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     3841 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
     3901 atgtggtatg gctgattatg atcctgcaag cctcgtcgcc gcggtttatt ctgttgacaa
     3961 ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat
     4021 gggatcggcc attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     4081 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     4141 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     4201 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     4261 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     4321 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     4381 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     4441 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     4501 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     4561 gcccggcggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     4621 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     4681 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     4741 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     4801 ccttcttgac gagttcttct gagcgggact ctggggttcg aataaagacc gaccaagcga
     4861 cgtctgagag ctccctggcg aattcggtac caataaaaga gctttatttt catgatctgt
     4921 gtgttggttt ttggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4981 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5041 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5101 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5161 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5221 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5281 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5341 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5401 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5461 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5521 cagaaaaaaa ggatctcaag aagatccttt gatcttttct agtgtgcg
//
