LOCUS       sequence-461648-        7233 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-GA donor plasmid for doxycycline-inducible expression of 
            jRCaMP1b calcium reporter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7233)
  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 7233)
  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_TRE-jRCaMP1b
FEATURES             Location/Qualifiers
     source          1..7233
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    55..88
                     /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(96..112)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(126..142)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(135..157)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      355..810
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(442..461)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     652..673
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(827..846)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     946..968
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1006..1024)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1092..1196
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1197..1265,1266..2057)
                     /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(1415..1434)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2228..2816
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2717..2736
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2970..2987
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3104..3125
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3140..3145,3146..3163,3164..3170)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3178..3194
                     /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     3183..3205
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3202..3218
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3202..3218
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3250..3460
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        3272..3468
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3319..3454
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3555..4733
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3786..4724
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4681..4701
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4741..4750
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4753..4790
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    4805..4852
                     /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    4927..4945
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4963..4981
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4999..5017
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5035..5053
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5071..5089
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5107..5125
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5143..5161
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     5206..5230
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5381..5398
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5402..5434
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(5438..5446,5447..5461)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(6786..6805)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6851..6906
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6905..6924)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
ORIGIN
        1 gaaaggacga tcggctgcaa gaactcttcc tcacggatcc gaagttccta ttccgaagtt
       61 cctattcttc aaatagtata ggaacttcag cgctcactgg ccgtcgtttt acaaagggcg
      121 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
      181 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
      241 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt
      301 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
      361 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      421 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      481 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      541 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
      601 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      661 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
      721 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
      781 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
      841 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
      901 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
      961 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     1021 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     1081 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1141 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     1201 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     1261 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     1321 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     1381 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     1441 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     1501 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     1561 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     1621 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     1681 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     1741 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     1801 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     1861 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     1921 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     1981 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     2041 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     2101 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     2161 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2221 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2281 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2341 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     2401 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2461 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2521 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2581 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2641 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     2701 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2761 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2821 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2881 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2941 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     3001 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     3061 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     3121 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     3181 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcttaatt
     3241 ccctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3301 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3361 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     3421 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctttgcaaa
     3481 gatggataaa gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga
     3541 aaggagtggg aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
     3601 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
     3661 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
     3721 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
     3781 cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt
     3841 gcgtgccttg aattacttcc acctggctgc agtacgtgat tcttgatccc gagcttcggg
     3901 ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt
     3961 gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg
     4021 cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga
     4081 cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt
     4141 cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga
     4201 ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc
     4261 ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg
     4321 cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga
     4381 gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga
     4441 aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt
     4501 ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg
     4561 ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt
     4621 ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc
     4681 tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaggaattc
     4741 gccaccatgg ccggcttgtc gacgacggcg gactccgtcg tcaggatcat ccggatccgg
     4801 atccgaagtt cctattccga agttcctatt ctctagaaag tataggaact tcatgatatt
     4861 cggcaagcag gcatcgacta gttaattagg aggcaagcag gcatcgacta gttaattaag
     4921 gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc agtgatagag
     4981 aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt ttactcccta
     5041 tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa cgtatctaca
     5101 gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc agtgatagag
     5161 aacgtataag ctttaggcgt gtacggtggg cgcctataaa agcagagctc gtttagtgaa
     5221 ccgtcagatc gcctggagca attccacaac acttttgtct tataccaact ttccgtacca
     5281 cttcctaccc tcgtaaactt aagaattcgc caccatgctg cagaacgagc ttgctcttaa
     5341 gttggctgga cttgatatta acaagactgg aggaggttct catcatcatc atcatcatgg
     5401 tatggctagc atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa
     5461 ggatctcgca acaatggtcg actcatcgcg acgtaagtgg aataagtggg gtcacgcagt
     5521 cagagctata ggtcggctga gctcagcgaa caacaccgaa atgatgtacc cagcggatgg
     5581 tggtctgcgt ggttacactc acatggcgct gaaagttgat ggcggcggtc acctgtcctg
     5641 ttctttcgtg accacctacc gctccaaaaa gactgtcggc aacattaaga tgcctgccat
     5701 tcattacgtc agccaccgtc tggagcgcct ggaggagagc gataacgaaa tgtttgtcgt
     5761 acagcgtgaa cacgcagttg ccaagtttgt gggcctgggt ggtggcggcg gtaccggagg
     5821 gagcatgaac tccctgatca aggagaacat gcgtatgaaa gtggttctgg aaggctccgt
     5881 aaacggccac cagttcaaat gcactggtga aggcgaaggc aacccgtata tgggcaccca
     5941 gactatgcgt atcaaagtga tcgagggtgg tccgctgccg tttgcgttcg acatcctggc
     6001 gacgtccttt atgtatggct cccgtacctt catcaaatat ccgaaaggca tcccggattt
     6061 ctttaagcag tccttcccgg aaggttttac ctgggaacgt gtgacccgtt acgaggacgg
     6121 cggcgtaatt accgttatgc aagacacgtc tctggaggat ggctgcctgg tgtatcacgt
     6181 gcaggttcgc ggtgtgaact tcccgagcaa tggtgctgta atgcaaaaga aaaccaaagg
     6241 ttgggagcct acggactccc aactgactga agagcagatc gcagaattta aagaggcttt
     6301 ctccctattt gacaaggacg gggatgggac aataacaacc aaggagatgg ggacggtgat
     6361 gcggtccctg gggcagaacc ccacagaagc agagctgcag gacatgatca atgaagtaga
     6421 tgccgacggt gacggcacaa tcgacttccc tgagttcctg attatgatgg caggcaaaat
     6481 gaaatacaca gacagtgaag aagaaattag agaagcgttc ggcgtgtttg ataaggatgg
     6541 caatggctac atcagtgcag cagagcttcg ccacgtgatg acaaaccttg gagagaagtt
     6601 aacagatgaa gaggttgatg aaatgatcag ggaagcagac agcgatgggg atggtcaggt
     6661 aaactacgaa gagtttgtac aaatgatgac agcgaagtag gtaaattcac tcctcaggtg
     6721 caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac
     6781 tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     6841 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     6901 ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg
     6961 gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa
     7021 agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc
     7081 ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc
     7141 ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca
     7201 gtcatagctg tccctcttct cttatgaaga tcg
//
