LOCUS       sequence-485733-        7971 bp    DNA     circular SYN 10-SEP-2025
DEFINITION  Bxb1-GT donor plasmid with CAG promoter, WPRE and polyA signal for 
            constitutive transgene expression (empty).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7971)
  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 7971)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 10, 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-GT-CAG Donor
FEATURES             Location/Qualifiers
     source          1..7971
                     /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     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        253..463
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        275..471
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      322..457
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        558..1736
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          789..1727
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1684..1704
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1744..1753
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1756..1793
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    complement(1802..1835)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        1945..2248
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          2570..3586
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3510..3532
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3594..3613
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      3647..3656
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3651..3660
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3664..3685
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3700..3705,3706..3723,3724..3730)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3738..3754
                     /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     3762..3778
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3774..4097
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    3791..3847
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3848..3864)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    4163..4751
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4216..4236)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4634..4645)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4804..5028
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(5060..5092)
                     /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(5100..5116)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      5329..5784
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5416..5435)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5626..5647
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5920..5942
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5980..5998)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6066..6170
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6171..6239,6240..7031)
                     /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(6389..6408)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7202..7790
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7691..7710
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7944..7961
                     /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 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
      421 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctttgc
      481 aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct
      541 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
      601 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
      661 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
      721 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
      781 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc
      841 cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc
      901 gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg
      961 cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc
     1021 gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg
     1081 cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta
     1141 tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg
     1201 cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc
     1261 ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc
     1321 tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag
     1381 ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa
     1441 ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc
     1501 cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg
     1561 aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag
     1621 cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca
     1681 ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaggaa
     1741 ttcgccacca tggccggctt gtcgacgacg gcggtctccg tcgtcaggat catccggatc
     1801 cataacttcg tatagcatac attatacgaa gttatcatga tattcggcaa gcaggcatcg
     1861 actagttaat taagtactta ctactagtta ttaatagtaa tcaattacgg ggtcattagt
     1921 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     1981 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     2041 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     2101 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     2161 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     2221 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
     2281 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
     2341 tgcagcgatg ggggcggggg gggggggggg gggggccccg gggggggggg gggggggggg
     2401 agggggggcg gggggggggg gggggggcgg gggggggggg ggggcggggg gggggggggg
     2461 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
     2521 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
     2581 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
     2641 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
     2701 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
     2761 gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg
     2821 cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg
     2881 cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc
     2941 gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg
     3001 gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg
     3061 agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct
     3121 cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg
     3181 ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg
     3241 cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc
     3301 tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg
     3361 gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc
     3421 gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg
     3481 ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc
     3541 tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt
     3601 gctggttatt gtgctgtctc atcattttgg caaaaatggc caccatgcca ccatggcggc
     3661 cgctaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     3721 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     3781 tgattacgcc aagcttgcat gcctgcaggt cgactctaga ggatccccgg gtaccgagct
     3841 cgaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     3901 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     3961 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt
     4021 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct
     4081 gctctgatgc cgcatagccc agcccgccta atgagcgggc ttttttttga acaaattaag
     4141 cctataacgc ggaagcggcc gcaatcaacc tctggattac aaaatttgtg aaagattgac
     4201 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     4261 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     4321 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     4381 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     4441 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     4501 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct
     4561 gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     4621 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     4681 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     4741 cgcctccccg ccctttgggc cgcctccccg cgctagtgct ggggatgcgt aacaacgttg
     4801 cacctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     4861 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     4921 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     4981 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggcg ctcgctcgct
     5041 gcaagaactc ttcctcacga taacttcgta taggagactt tatacgaagt taagcgctca
     5101 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     5161 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     5221 ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt
     5281 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     5341 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     5401 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     5461 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     5521 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     5581 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     5641 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     5701 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     5761 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     5821 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5881 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5941 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     6001 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     6061 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     6121 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     6181 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     6241 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     6301 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     6361 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     6421 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     6481 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6541 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     6601 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6661 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6721 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6781 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6841 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6901 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6961 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     7021 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     7081 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     7141 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     7201 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     7261 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     7321 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     7381 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     7441 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     7501 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7561 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     7621 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7681 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7741 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7801 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     7861 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     7921 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g
//
