LOCUS       sequence-362566-        4813 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-specific donor plasmid for the cloning of payloads.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4813)
  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 4813)
  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)_lox
FEATURES             Location/Qualifiers
     source          1..4813
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    110..131
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(146..151,152..169,170..176)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    184..200
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     189..211
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     208..224
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     208..224
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        256..466
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        278..474
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      325..460
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        561..1739
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          792..1730
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1687..1707
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1747..1756
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1759..1796
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    complement(1805..1838)
                     /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(1905..1937)
                     /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(1945..1962)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1945..1961)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1954..1976)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2174..2629
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2261..2280)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2471..2492
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2646..2665)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2765..2787
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2825..2843)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2911..3015
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3016..3084,3085..3876)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3234..3253)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4047..4635
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4536..4555
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4789..4806
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
       61 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
      121 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
      181 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
      241 ttaattccct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
      301 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
      361 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
      421 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt
      481 tgcaaagatg gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg
      541 tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
      601 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
      661 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
      721 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
      781 ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg
      841 gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc
      901 ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc
      961 gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc
     1021 ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg
     1081 ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg
     1141 gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt
     1201 cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg
     1261 gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa
     1321 ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg
     1381 cagggagctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac
     1441 aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg
     1501 cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg
     1561 gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc
     1621 cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt
     1681 tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgag
     1741 gaattcgcca ccatggccgg cttgtcgacg acggcggtct ccgtcgtcag gatcatccgg
     1801 atccataact tcgtatagca tacattatac gaagttatca tgatattcgg caagcaggca
     1861 tcgactagtt aattaagcta gcgctgcaag aactcttcct cacgataact tcgtatagga
     1921 gactttatac gaagttaagc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
     1981 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     2041 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     2101 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     2161 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2221 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     2281 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2341 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     2401 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2461 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     2521 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     2581 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     2641 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     2701 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     2761 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     2821 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     2881 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2941 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3001 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     3061 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3121 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3181 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     3241 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     3301 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3361 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3421 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3481 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3541 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3601 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3661 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3721 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3781 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3841 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3901 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3961 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4021 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4081 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4141 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     4201 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4261 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4321 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4381 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4441 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4501 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4561 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4621 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4681 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4741 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4801 agcgaggaag cgg
//
