LOCUS       sequence-461682-        6552 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-GT donor plasmid for doxycycline-inducible mScarlet expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6552)
  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 6552)
  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-GT_TRE-mScarlet
FEATURES             Location/Qualifiers
     source          1..6552
                     /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"
     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)."
     protein_bind    1881..1899
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1917..1935
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1953..1971
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1989..2007
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2025..2043
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2061..2079
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2097..2115
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2160..2184
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2263..2272
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2269..2964
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2402..2420)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2670..2689
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2968..3556
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3021..3041)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3439..3450)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(3641..3673)
                     /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(3681..3698)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3681..3697)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3690..3712)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3910..4365
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3997..4016)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4207..4228
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4382..4401)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4501..4523
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4561..4579)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4647..4751
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4752..4820,4821..5612)
                     /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(4970..4989)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5783..6371
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6272..6291
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6525..6542
                     /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 taaggtttac tccctatcag tgatagagaa cgtatgaaga gtttactccc
     1921 tatcagtgat agagaacgta tgcagacttt actccctatc agtgatagag aacgtataag
     1981 gagtttactc cctatcagtg atagagaacg tatgaccagt ttactcccta tcagtgatag
     2041 agaacgtatc tacagtttac tccctatcag tgatagagaa cgtatatcca gtttactccc
     2101 tatcagtgat agagaacgta taagctttag gcgtgtacgg tgggcgccta taaaagcaga
     2161 gctcgtttag tgaaccgtca gatcgcctgg agcaattcca caacactttt gtcttatacc
     2221 aactttccgt accacttcct accctcgtaa acttaagaat tcgccaccat ggtgagcaag
     2281 ggcgaggcag tgatcaagga gttcatgcgg ttcaaggtgc acatggaggg ctccatgaac
     2341 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     2401 gccaagctga aggtgaccaa gggtggcccc ctgcccttct cctgggacat cctgtcccct
     2461 cagttcatgt acggctccag ggccttcacc aagcaccccg ccgacatccc cgactactat
     2521 aagcagtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     2581 gccgtgaccg tgacccagga cacctccctg gaggacggca ccctgatcta caaggtgaag
     2641 ctccgcggca ccaacttccc tcctgacggc cccgtaatgc agaagaagac aatgggctgg
     2701 gaagcgtcca ccgagcggtt gtaccccgag gacggcgtgc tgaagggcga cattaagatg
     2761 gccctgcgcc tgaaggacgg cggccgctac ctggcggact tcaagaccac ctacaaggcc
     2821 aagaagcccg tgcagatgcc cggcgcctac aacgtcgacc gcaagttgga catcacctcc
     2881 cacaacgagg actacaccgt ggtggaacag tacgaacgct ccgagggccg ccactccacc
     2941 ggcggcatgg acgagctgta caagtaaaat caacctctgg attacaaaat ttgtgaaaga
     3001 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     3061 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     3121 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     3181 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     3241 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     3301 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     3361 aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     3421 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     3481 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     3541 tgggccgcct ccccgccctt tgggccgcct ccccgcgcta gtgctgggga tgcggtgggc
     3601 tctatgggtc tcgacattgc tgcaagaact cttcctcacg ataacttcgt ataggagact
     3661 ttatacgaag ttaagcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     3721 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     3781 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     3841 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     3901 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3961 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     4021 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4081 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     4141 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4201 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     4261 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     4321 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     4381 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     4441 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4501 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     4561 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     4621 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4681 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4741 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4801 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4861 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4921 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4981 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5041 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5101 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5161 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5221 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     5281 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     5341 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5401 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5461 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5521 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5581 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5641 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5701 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5761 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5821 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5881 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5941 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6001 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6061 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6121 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6181 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6241 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6301 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6361 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6421 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6481 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     6541 aggaagcgga ag
//
