LOCUS       sequence-370598-        6075 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-specific donor plasmid for the cloning of multiple inserts by 
            Golden Gate assembly (BpiI).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6075)
  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 6075)
  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: pBR_attB(bxb)_ccdB_lox
FEATURES             Location/Qualifiers
     source          1..6075
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(82..1230)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(1566..2154)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1646..1665)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2325..3119)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     promoter        complement(3120..3224)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3245..3955
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4043..4067
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     4103..4120
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4104..4120
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4128..4160
                     /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)."
     CDS             complement(4265..4570)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(4367..4386)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    4634..4667
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4676..4713
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      4716..4725
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4733..5911)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4742..5680
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(4765..4785)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    5987..6011
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 catcaaccat gactactgtc cggtccaaca gatccgagca ggggctgctg aacccccagc
       61 cggaactgac cccacaaggc cctagcgttt gcaatgcacc aggtcatcat tgacccaggc
      121 gtgttccacc aggccgctgc ctcgcaactc ttcgcaggct tcgccgacct gctcgcgcca
      181 cttcttcacg cgggtggaat ccgatccgca catgaggcgg aaggtttcca gcttgagcgg
      241 gtacggctcc cggtgcgagc tgaaatagtc gaacatccgt cgggccgtcg gcgacagctt
      301 gcggtacttc tcccatatga atttcgtgta gtggtcgcca gcaaacagca cgacgatttc
      361 ctcgtcgatc aggacctggc aacgggacgt tttcttgcca cggtccagga cgcggaagcg
      421 gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcg gacgtgaagc ccatcgccgt
      481 cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaa taccggccat tgatcgacca
      541 gcccaggtcc tggcaaagct cgtagaacgt gaaggtgatc ggctcgccga taggggtgcg
      601 cttcgcgtac tccaacacct gctgccacac cagttcgtca tcgtcggccc gcagctcgac
      661 gccggtgtag gtgatcttca cgtccttgtt gacgtggaaa atgaccttgt tttgcagcgc
      721 ctcgcgcggg attttcttgt tgcgcgtggt gaacagggca gagcgggccg tgtcgtttgg
      781 catcgctcgc atcgtgtccg gccacggcgc aatatcgaac aaggaaagct gcatttcctt
      841 gatctgctgc ttcgtgtgtt tcagcaacgc ggcctgcttg gcctcgctga cctgttttgc
      901 caggtcctcg ccggcggttt ttcgcttctt ggtcgtcata gttcctcgcg tgtcgatggt
      961 catcgacttc gccaaacctg ccgcctcctg ttcaagacga cgcgaacgct ccacggcggc
     1021 cgatggcgcg ggcagggcag ggggagccag ttgcacgctg tcgcgctcga tcttggccgt
     1081 agcttgctgg accatcgagc cgacggactg gaaggtttcg cggggcgcac gcatgacggt
     1141 gcggcttgcg atggtttcgg catcctcggc ggaaaacccc gcgtcgatca gttcttgcct
     1201 gtatgccttc cggtcaaacg tccgattcat tcaccctcct tgcgggattg ccccgactca
     1261 cgccggggca atgtgccctt attcctgatt tgacccgcct ggtgccttgg tgtccagata
     1321 atccacctta tcggcaatga agtcggtccc gtagaccgtc tggccgtcct tctcgtactt
     1381 ggtattccga atcttgccct gcacgaatac cagcgacccc ttgcccaaat acttgccgtg
     1441 ggcctcggcc tgagagccaa aacacttgat gcggaagaag tcggtgcgct cctgcttgtc
     1501 gccggcatcg ttgcgccaca tctaggatct gccaggaacc gtaaaaaggc cgcgttgctg
     1561 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     1621 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1681 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1741 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     1801 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1861 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     1921 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     1981 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     2041 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2101 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2161 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     2221 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     2281 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagctaaaa caattcatcc
     2341 agtaaaatat aatattttat tttctcccaa tcaggcttga tccccagtaa gtcaaaaaat
     2401 agctcgacat actgttcttc cccgatatcc tccctgagcg accggacgca gaaggcaatg
     2461 tcataccact tgtccgccct gccgcttctc ccaagatcaa taaagccact tactttgcca
     2521 tctttcacaa agatgttgct gtctcccagg tcgccgtggg aaaagacaag ttcctcttcg
     2581 ggcttttccg tctttaaaaa atcatacagc tcgcgcggat ctttaaatgg agtgtcctct
     2641 tcccagtttt cgcaatccac atcggccaga tcgttattca gtaagtaatc caattcggct
     2701 aagcggctgt ctaagctatt cgtataggga caatccgata tgtcgatgga gtgaaagagc
     2761 ctgatgcact ccgcatacag ctcgataatc ttttcagggc tttgttcatc ttcatactct
     2821 tccgagcaaa ggacgccatc ggcctcactc atgagcagat tgctccagcc atcatgccgt
     2881 tcaaagtgca ggacctttgg aacaggcagc tttccttcca gccatagcat catgtccttt
     2941 tcccgttcca catcataggt ggtcccttta taccggctgt ccgtcatttt taaatatagg
     3001 ttttcatttt ctcccaccag cttatatacc ttagcaggag acattccttc cgtatctttt
     3061 acgcagcggt atttttcgat cagttttttc aattccggtg atattctcat tttagccata
     3121 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3181 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcactcg ggaattggcc
     3241 agcgctgcca tttttggggt gaggccgttc gcggccgagg ggcgcagccc ctggggggat
     3301 gggaggcccg cgttagcggg ccgggagggt tcgagaaggg ggggcacccc ccttcggcgt
     3361 gcgcggtcac gcgcacaggg cgcagccctg gttaaaaaca aggtttataa atattggttt
     3421 aaaagcaggt taaaagacag gttagcggtg gccgaaaaac gggcggaaac ccttgcaaat
     3481 gctggatttt ctgcctgtgg acagcccctc aaatgtcaat aggtgcgccc ctcatctgtc
     3541 agcactctgc ccctcaagtg tcaaggatcg cgcccctcat ctgtcagtag tcgcgcccct
     3601 caagtgtcaa taccgcaggg cacttatccc caggcttgtc cacatcatct gtgggaaact
     3661 cgcgtaaaat caggcgtttt cgccgatttg cgaggctggc cagctccacg tcgccggccg
     3721 aaatcgagcc tgcccctcat ctgtcaacgc cgcgccgggt gagtcggccc ctcaagtgtc
     3781 aacgtccgcc cctcatctgt cagtgagggc caagttttcc gcgaggtatc cacaacgccg
     3841 gcggccgcgg tgtctcgcac acggcttcga cggcgtttct ggcgcgtttg cagggccata
     3901 gacggccgcc agcccagcgg cgagggcaac cagcccggtg agcgtcgcaa aggcggctgg
     3961 cacatacaaa tggacgaacg gataaacctt ttcacgccct tttaaatatc cgattattct
     4021 aataaacgct cttttctctt aggtttaccc gccaatatat cctgtcaaac actgatagtt
     4081 taaacggatc acttcgtgtc cctgtaaaac gacggccagt gagcgcttaa cttcgtataa
     4141 agtctcctat acgaagttat cgtgaggaag agttcttgca gctcctccca tgtcttctga
     4201 agtcagcccc atacgatata agttgttttg ttcaaaaaaa agcccgctca ttaggcgggc
     4261 tgggttatat tccccagaac atcaggttaa tggcgttttt gatgtcattt tcgcggtggc
     4321 tgagatcagc cacttcttcc ccgataacgg agaccggcac actggccata tcggtggtca
     4381 tcatgcgcca gctttcatcc ccgatatgca ccaccgggta aagttcacgg gagactttat
     4441 ctgacagcag acgtgcactg gccaggggga tcaccatccg tcgcccgggc gtgtcaataa
     4501 tatcactctg tacatccaca aacagacgat aacggctctc tcttttatag gtgtaaacct
     4561 taaactgcat aatctgacct cctggttatg tggaagacta ggcagctacg atgcctgctt
     4621 gccgaatatc atgataactt cgtataatgt atgctatacg aagttatgga tccggatgat
     4681 cctgacgacg gagaccgccg tcgtcgacaa gccggccatg gtggcgaatt cctcacgaca
     4741 cctgaaatgg aagaaaaaaa ctttgaacca ctgtctgagg cttgagaatg aaccaagatc
     4801 caaactcaaa aagggcaaat tccaaggaga attacatcaa gtgccaagct ggcctaactt
     4861 cagtctccac ccactcagtg tggggaaact ccatcgcata aaacccctcc ccccaaccta
     4921 aagacgacgt actccaaaag ctcgagaact aatcgaggtg cctggacggc gcccggtact
     4981 ccgtggagtc acatgaagcg acggctgagg acggaaaggc ccttttcctt tgtgtgggtg
     5041 actcacccgc ccgctctccc gagcgccgcg tcctccattt tgagctccct gcagcagggc
     5101 cgggaagcgg ccatctttcc gctcacgcaa ctggtgccga ccgggccagc cttgccgccc
     5161 agggcggggc gatacacggc ggcgcgaggc caggcaccag agcaggccgg ccagcttgag
     5221 actacccccg tccgattctc ggtggccgcg ctcgcaggcc ccgcctcgcc gaacatgtgc
     5281 gctgggacgc acgggccccg tcgccgcccg cggccccaaa aaccgaaata ccagtgtgca
     5341 gatcttggcc cgcatttaca agactatctt gccagaaaaa aagcgtcgca gcaggtcatc
     5401 aaaaatttta aatggctaga gacttatcga aagcagcgag acaggcgcga aggtgccacc
     5461 agattcgcac gcggcggccc cagcgcccag gccaggcctc aactcaagca cgaggcgaag
     5521 gggctcctta agcgcaaggc ctcgaactct cccacccact tccaacccga agctcgggat
     5581 caagaatcac gtactgcagc caggtggaag taattcaagg cacgcaaggg ccataacccg
     5641 taaagaggcc aggcccgcgg gaaccacaca cggcacttac ctgtgttctg gcggcaaacc
     5701 cgttgcgaaa aagaacgttc acggcgacta ctgcacttat atacggttct cccccaccct
     5761 cgggaaaaag gcggagccag tacacgacat cactttccca gtttaccccg cgccaccttc
     5821 tctaggcacc ggttcaattg ccgacccctc cccccaactt ctcggggact gtgggcgatg
     5881 tgcgctctgc ccactgacgg gcaccggagc caatggcaca gagtgttcaa ccccagtaca
     5941 ttaaaaacgt ccgcaatgtg ttattaagtt gtctaagcgt caatttgttt acaccacaat
     6001 atatcctgcc accagccagc caacagctcc ccgaccggca gctcggcaca aaatcaccac
     6061 tcgatacagg cagcc
//
