LOCUS       sequence-365730-        3758 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-specific donor plasmid for the cloning of payloads (~10 - 50 
            kb).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3758)
  AUTHORS   Blanch-Asensio A, Grandela C, Brandao KO, de Korte T, Mei H, 
            Ariyurek Y, Yiangou L, Mol MPH, van Meer BJ, Kloet SL, Mummery CL, 
            Davis RP
  TITLE     STRAIGHT-IN enables high-throughput targeting of large DNA payloads 
            in human pluripotent stem cells.
  JOURNAL   Cell Rep Methods. 2022 Sep 22;2(10):100300. doi: 
            10.1016/j.crmeth.2022.100300. eCollection 2022 Oct 24.
  PUBMED    36313798
REFERENCE   2  (bases 1 to 3758)
  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: "Cell Rep 
            Methods."; date: "2022-09-22"; volume: "2(10):100300. doi"; pages: "
            10.1016/j.crmeth.2022.100300. eCollection 2022 Oct 24"
COMMENT     Sequence Label: p15_attB(bxb)_lox
FEATURES             Location/Qualifiers
     source          1..3758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        64..1242
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          295..1233
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1190..1210
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1250..1259
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1262..1299
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    complement(1308..1341)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1408..1440)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(1448..1465)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1448..1464)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1457..1479)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        1596..1700
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1701..1769,1770..2561)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1919..1938)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2726..3271
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     3172..3191
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3388..3405
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3539..3735
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3586..3721
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa aggagtggga
       61 attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg
      121 gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa
      181 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
      241 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt
      301 gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga
      361 attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg
      421 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc
      481 tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct
      541 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc
      601 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg
      661 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg
      721 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg
      781 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca
      841 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg
      901 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt
      961 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc
     1021 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg
     1081 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg
     1141 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag
     1201 acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggaattcg ccaccatggc
     1261 cggcttgtcg acgacggcgg tctccgtcgt caggatcatc cggatccata acttcgtata
     1321 gcatacatta tacgaagtta tcatgatatt cggcaagcag gcatcgacta gttaattaag
     1381 ctagcgctgc aagaactctt cctcacgata acttcgtata ggagacttta tacgaagtta
     1441 agcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     1501 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     1561 ccgatacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1621 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1681 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1741 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1801 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1861 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1921 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     1981 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2041 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2101 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2161 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2221 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     2281 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2341 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2401 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2461 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2521 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2581 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2641 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2701 cagacccctt aataagatga tcttcttgag atcgttttgg tctgcgcgta atctcttgct
     2761 ctgaaaacga aaaaaccgcc ttgcagggcg gtttttcgaa ggttctctga gctaccaact
     2821 ctttgaaccg aggtaactgg cttggaggag cgcagtcacc aaaacttgtc ctttcagttt
     2881 agccttaacc ggcgcatgac ttcaagacta actcctctaa atcaattacc agtggctgct
     2941 gccagtggtg cttttgcatg tctttccggg ttggactcaa gacgatagtt accggataag
     3001 gcgcagcggt cggactgaac ggggggttcg tgcatacagt ccagcttgga gcgaactgcc
     3061 tacccggaac tgagtgtcag gcgtggaatg agacaaacgc ggccataaca gcggaatgac
     3121 accggtaaac cgaaaggcag gaacaggaga gcgcacgagg gagccgccag ggggaaacgc
     3181 ctggtatctt tatagtcctg tcgggtttcg ccaccactga tttgagcgtc agatttcgtg
     3241 atgcttgtca ggggggcgga gcctatggaa aaacggcttt gccgcggccc tctcacttcc
     3301 ctgttaagta tcttcctggc atcttccagg aaatctccgc cccgttcgta agccatttcc
     3361 gctcgccgca gtcgaacgac cgagcgtagc gagtcagtga gcgaggaagc ggaatatatc
     3421 ctgtatcaca tattctgctg acgcaccggt gcagcctttt ttctcctgcc acatgaagca
     3481 cttcactgac accctcatca gtgccaacat agtaagccag tatacactcc gctagcgctg
     3541 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3601 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3661 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3721 ctaggctttt gcaaaaagct ttgcaaagat ggataaag
//
