LOCUS       sequence-489343-        4304 bp    DNA     circular SYN 15-OCT-2025
DEFINITION  Bxb1-GA donor plasmid for cloning of payloads.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4304)
  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 4304)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 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_CBh
FEATURES             Location/Qualifiers
     source          1..4304
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        363..648
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        650..926
                     /label=chicken beta-actin promoter
     intron          927..1154
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1166..1175
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1178..1215
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    1230..1277
                     /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)."
     primer_bind     complement(1403..1419)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1433..1449)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1442..1464)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1662..2117
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1749..1768)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1959..1980
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2134..2153)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2253..2275
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2313..2331)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2399..2503
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2504..2572,2573..3364)
                     /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(2722..2741)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3535..4123
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4024..4043
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4277..4294
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctta
      241 attccctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
      301 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
      361 cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      421 ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      481 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      541 gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac cttatgggac
      601 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc
      661 cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta
      721 tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg cgcgcgccag
      781 gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca
      841 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
      901 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc
      961 gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg
     1021 tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt
     1081 ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa
     1141 tcactttttt tcaggttgga ccggtgccac catggccggc ttgtcgacga cggcggactc
     1201 cgtcgtcagg atcatccgga tccggatccg aagttcctat tccgaagttc ctattctcta
     1261 gaaagtatag gaacttcatg atattcggca agcaggcatc gactagttaa ttaagctagc
     1321 gctgcaagaa ctcttcctca cggatccgaa gttcctattc cgaagttcct attcttcaaa
     1381 tagtatagga acttcagcgc tcactggccg tcgttttaca aagggcgaat tcactggccg
     1441 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     1501 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     1561 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     1621 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     1681 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     1741 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     1801 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1861 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1921 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1981 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     2041 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     2101 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     2161 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     2221 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     2281 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     2341 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     2401 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2461 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2521 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2581 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2641 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2701 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     2761 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2821 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     2881 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     2941 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3001 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3061 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3121 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3181 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     3241 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3301 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3361 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3421 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3481 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3541 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3601 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3661 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     3721 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3781 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3841 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3901 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3961 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4021 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4081 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4141 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4201 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4261 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaag
//
