LOCUS       sequence-450363-        4503 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1 payload vector with 5' attP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4503)
  AUTHORS   Fell CW, Schmitt-Ulms C, Tagliaferri DV, Gootenberg JS, Abudayyeh OO
  TITLE     Precise kilobase-scale genomic insertions in mammalian cells using 
            PASTE.
  JOURNAL   Nat Protoc. 2024 Dec 15. doi: 10.1038/s41596-024-01090-z.
  PUBMED    39676077
REFERENCE   2  (bases 1 to 4503)
  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 Protoc.
            2024 Dec 15. doi: 10.1038/s41596-024-01090-z."
COMMENT     Sequence Label: PASTE payload acceptor with attP site pDY2087
FEATURES             Location/Qualifiers
     source          1..4503
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(39..58)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     649..668
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(1086..1103)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1256..1342
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1434..1461
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      1595..2183
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2084..2103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2337..2354
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2369..2509
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2435..2454)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2595..2617
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3296..3313)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    3334..3367
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     protein_bind    3415..3448
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3466..3513
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             3532..4197
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     CDS             3532..4197
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     primer_bind     4270..4289
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(4431..4503,1..722))
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(join(4431..4503,1..722))
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
ORIGIN
        1 aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct
       61 atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg
      121 ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg
      181 ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc
      241 tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg
      301 atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc
      361 cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga
      421 tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg
      481 agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct
      541 tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc
      601 gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag
      661 ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc
      721 atgcgaaacg atcctcatcc tgtctcttga tcagagcttg atcccctgcg ccatcagatc
      781 cttggcggca agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc
      841 gccccagctg gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc
      901 catgtaagcc cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag
      961 atagcccagt agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac
     1021 gcgctcgagg gggggccaaa cggtctccag cttggctgtt ttggcggatg agagaagatt
     1081 ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct
     1141 ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt
     1201 agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat
     1261 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     1321 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
     1381 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc
     1441 catcctgacg gatggccttt ttgcgtttct acaaactctt ttgtttattt ttctaaatac
     1501 attcaaatat gtatccgctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     1561 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     1621 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     1681 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     1741 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     1801 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     1861 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     1921 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     1981 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     2041 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2101 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2161 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     2221 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     2281 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     2341 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     2401 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     2461 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     2521 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     2581 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     2641 cgcgaggcag cagatcaatt cgcgcgcgaa ggcgaagcgg catgcataat gtgcctgtca
     2701 aatggacgaa gcagggattc tgcaaaccct atgctactcc gtcaagccgt caattgtctg
     2761 attcgttacc aattatgaca acttgacggc tacatcattc actttttctt cacaaccggc
     2821 acggaactcg ctcgggctgg ccccggtgca ttttttaaat acccgcgaga aatagagttg
     2881 atcgtcaaaa ccaacattgc gaccgacggt ggcgataggc atccgggtgg tgctcaaaag
     2941 cagcttcgcc tggctgatac gttggtcctc gcgccagctt aagacgctaa tccctaactg
     3001 ctggcggaaa agatgtgaca gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc
     3061 gattcttctc tcatccgcca aaacagccaa gctggagacc gtttgacatt accctgttat
     3121 ccctagatac attaccctgt tatcccagat gacataccct gttatcccta gatgacatta
     3181 ccctgttatc ccagatgaca ttaccctgtt atccctagat acattaccct gttatcccag
     3241 atgacatacc ctgttatccc tagatgacat taccctgtta tcccagataa actcaatgat
     3301 gatgatgatg atggtcgaga ctcagcggcc gcggtgccag ggcgtgccct tgggctcccc
     3361 gggcgcgact ataagctgcg agcaacttca cttgggtatg ccggcggtag cgcttaccgt
     3421 tcgtataatg tatgctatac gaagttatcc gaagccgcta gcggtggttt gtctggtcaa
     3481 ccaccgcggt ctcagtggtg tacggtacaa acccagctac cggtcgccac catgcccgcc
     3541 atgaagatcg agtgccgcat caccggcacc ctgaacggcg tggagttcga gctggtgggc
     3601 ggcggagagg gcacccccga gcagggccgc atgaccaaca agatgaagag caccaaaggc
     3661 gccctgacct tcagccccta cctgctgagc cacgtgatgg gctacggctt ctaccacttc
     3721 ggcacctacc ccagcggcta cgagaacccc ttcctgcacg ccatcaacaa cggcggctac
     3781 accaacaccc gcatcgagaa gtacgaggac ggcggcgtgc tgcacgtgag cttcagctac
     3841 cgctacgagg ccggccgcgt gatcggcgac ttcaaggtgg tgggcaccgg cttccccgag
     3901 gacagcgtga tcttcaccga caagatcatc cgcagcaacg ccaccgtgga gcacctgcac
     3961 cccatgggcg ataacgtgct ggtgggcagc ttcgcccgca ccttcagcct gcgcgacggc
     4021 ggctactaca gcttcgtggt ggacagccac atgcacttca agagcgccat ccaccccagc
     4081 atcctgcaga acgggggccc catgttcgcc ttccgccgcg tggaggagct gcacagcaac
     4141 accgagctgg gcatcgtgga gtaccagcac gccttcaaga cccccatcgc cttcgccaga
     4201 tctcgactcg agctcgatga gtctagacaa accacaacta gaatgcagtg aaaaaaatgc
     4261 tttatttgtg aaatttgtga tgctattgct ttatttgtgg gcccgcccca actggggtaa
     4321 cctttgagtt ctctcagttg ggggtaatca gcatcatgat gtggtaccac atcatgatgc
     4381 tgattataag aatgcggccg ccacactcta gtggatctcg agttaataat tcagaagaac
     4441 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc
     4501 acg
//
