LOCUS       sequence-461911-        5373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-GA donor plasmid for the cloning of payloads.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5373)
  AUTHORS   Blanch-Asensio A, Ploessl DS, Wang NB, Mummery CL, Galloway KE, 
            Davis RP
  TITLE     STRAIGHT-IN Dual: a platform for dual, single-copy integrations of 
            DNA payloads and gene circuits into human induced pluripotent stem 
            cells.
  JOURNAL   bioRxiv [Preprint]. 2024 Oct 17:2024.10.17.616637. doi: 
            10.1101/2024.10.17.616637.
  PUBMED    39464154
REFERENCE   2  (bases 1 to 5373)
  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: "bioRxiv 
            [Preprint]. 2024 Oct 17:2024.10.17.616637. doi: 
            10.1101/2024.10.17.616637."
COMMENT     Sequence Label: Bxb1-GA Donor_CAG
FEATURES             Location/Qualifiers
     source          1..5373
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    9..46
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    61..108
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    193..226
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     primer_bind     complement(234..250)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(264..280)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(273..295)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      493..948
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(580..599)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     790..811
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(965..984)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1084..1106
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1144..1162)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1230..1334
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1335..1403,1404..2195)
                     /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(1553..1572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2366..2954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2855..2874
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3108..3125
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3242..3263
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3278..3283,3284..3301,3302..3308)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3316..3332
                     /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     3321..3343
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3340..3356
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3340..3356
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3388..3598
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        3410..3606
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3457..3592
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        3712..4015
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4017..4291
                     /label=chicken beta-actin promoter
     intron          4292..5308
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     5232..5254
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     5316..5335
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      join(5370..5373,1..6)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ccatggccgg cttgtcgacg acggcggact ccgtcgtcag gatcatccgg atccggatcc
       61 gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcat gatattcggc
      121 aagcaggcat cgactagtta attaagctag cgctgcaaga actcttcctc acggatccga
      181 agttcctatt ccgaagttcc tattcttcaa atagtatagg aacttcagcg ctcactggcc
      241 gtcgttttac aaagggcgaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
      301 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
      361 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
      421 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
      481 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
      541 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
      601 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      661 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
      721 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
      781 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
      841 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
      901 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
      961 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     1021 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     1081 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     1141 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     1201 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1261 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1321 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1381 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1441 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1501 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1561 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1621 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1681 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1741 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1801 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1861 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1921 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1981 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2041 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2101 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2161 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2221 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2281 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2341 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2401 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2461 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2521 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2581 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2641 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2701 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2761 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2821 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2881 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2941 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3001 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3061 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3121 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     3181 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     3241 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     3301 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     3361 attacgccaa gcttaattcc ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3421 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3481 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3541 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3601 tgcaaaaagc tttgcaaaga tggataaagt tttaaacaga gaggaatctt tgcagctaat
     3661 ggaccttcta ggtcttgaaa ggagtgggaa ttggctccta ctggagttcc gcgttacata
     3721 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     3781 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     3841 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     3901 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     3961 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga
     4021 ggtgagcccc acgttctgct tcactctccc catctccccc cccccccccc ccccaatttt
     4081 gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg gggggcgcgc
     4141 gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc
     4201 agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg
     4261 gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt
     4321 gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc
     4381 acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg
     4441 acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg agggcccttt
     4501 gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg
     4561 cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct
     4621 ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg
     4681 cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg
     4741 cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg agcacggccc
     4801 ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg
     4861 gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg agggctcggg
     4921 ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca
     4981 ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc aaatctgtgc
     5041 ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg cgaagcggtg
     5101 cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc
     5161 ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc gggggggacg
     5221 gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca
     5281 tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc
     5341 tcatcatttt ggcaaagaat tccgatcgcg cca
//
