LOCUS       sequence-463599-        7487 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-GT donor plasmid for integrating a transcription unit for 
            constitutive expression of a mScarlet-I F-actin reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7487)
  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 7487)
  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_CAG-ITPKA-mScarlet-I
FEATURES             Location/Qualifiers
     source          1..7487
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    21..58
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      61..70
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(78..1256)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          87..1025
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(110..130)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(1343..1539)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(1351..1561)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1357..1492
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1593..1609)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1593..1609)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1606..1628)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1617..1633
                     /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)."
     promoter        complement(join(1641..1647,1648..1665,1666..1671))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1686..1707
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1824..1841)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1995..2583)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2075..2094)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2754..3545,3546..3614))
                     /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     3377..3396
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3615..3719)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3787..3805
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3843..3865)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3965..3984
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4001..4456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4138..4159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4350..4369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4654..4676
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4668..4685
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4669..4685
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4693..4725
                     /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)."
     polyA_signal    4751..4872
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4762..4781)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4816..4835
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4998..5693)
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(5273..5292)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5542..5560
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      5690..5699
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          5826..6842
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(5880..5902)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(6843..7116)
                     /label=chicken beta-actin promoter
     enhancer        7118..7421
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    join(7466..7487,1..12)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 atacgaagtt atggatccgg atgatcctga cgacggagac cgccgtcgtc gacaagccgg
       61 ccatggtggc gaattcctca cgacacctga aatggaagaa aaaaactttg aaccactgtc
      121 tgaggcttga gaatgaacca agatccaaac tcaaaaaggg caaattccaa ggagaattac
      181 atcaagtgcc aagctggcct aacttcagtc tccacccact cagtgtgggg aaactccatc
      241 gcataaaacc cctcccccca acctaaagac gacgtactcc aaaagctcga gaactaatcg
      301 aggtgcctgg acggcgcccg gtactccgtg gagtcacatg aagcgacggc tgaggacgga
      361 aaggcccttt tcctttgtgt gggtgactca cccgcccgct ctcccgagcg ccgcgtcctc
      421 cattttgagc tccctgcagc agggccggga agcggccatc tttccgctca cgcaactggt
      481 gccgaccggg ccagccttgc cgcccagggc ggggcgatac acggcggcgc gaggccaggc
      541 accagagcag gccggccagc ttgagactac ccccgtccga ttctcggtgg ccgcgctcgc
      601 aggccccgcc tcgccgaaca tgtgcgctgg gacgcacggg ccccgtcgcc gcccgcggcc
      661 ccaaaaaccg aaataccagt gtgcagatct tggcccgcat ttacaagact atcttgccag
      721 aaaaaaagcg tcgcagcagg tcatcaaaaa ttttaaatgg ctagagactt atcgaaagca
      781 gcgagacagg cgcgaaggtg ccaccagatt cgcacgcggc ggccccagcg cccaggccag
      841 gcctcaactc aagcacgagg cgaaggggct ccttaagcgc aaggcctcga actctcccac
      901 ccacttccaa cccgaagctc gggatcaaga atcacgtact gcagccaggt ggaagtaatt
      961 caaggcacgc aagggccata acccgtaaag aggccaggcc cgcgggaacc acacacggca
     1021 cttacctgtg ttctggcggc aaacccgttg cgaaaaagaa cgttcacggc gactactgca
     1081 cttatatacg gttctccccc accctcggga aaaaggcgga gccagtacac gacatcactt
     1141 tcccagttta ccccgcgcca ccttctctag gcaccggttc aattgccgac ccctcccccc
     1201 aacttctcgg ggactgtggg cgatgtgcgc tctgcccact gacgggcacc ggagccaatt
     1261 cccactcctt tcaagaccta gaaggtccat tagctgcaaa gattcctctc tgtttaaaac
     1321 tttatccatc tttgcaaagc tttttgcaaa agcctaggcc tccaaaaaag cctcctcact
     1381 acttctggaa tagctcagag gccgaggcgg cctcggcctc tgcataaata aaaaaaatta
     1441 gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag
     1501 ttaggggcgg gactatggtt gctgactaat tgagatgcat gctttgcata cttctgcctg
     1561 ctggggaggg aattaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     1621 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     1681 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     1741 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     1801 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     1861 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     1921 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     1981 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     2041 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     2101 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     2161 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     2221 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     2281 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     2341 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     2401 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     2461 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     2521 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     2581 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     2641 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     2701 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     2761 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     2821 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     2881 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     2941 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3001 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3061 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3121 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3181 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3241 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3301 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3361 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3421 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3481 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     3541 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     3601 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     3661 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     3721 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     3781 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     3841 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     3901 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     3961 tatgcggcat cagagcagat tgtactgaga gtgcaccata aaattgtaaa cgttaatatt
     4021 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca atagaccgaa
     4081 atcggcaaaa tcccttataa atcaaaagaa tagcccgaga tagagttgag tgttgttcca
     4141 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     4201 gtctatcagg gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttggggtcg
     4261 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     4321 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctaag
     4381 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     4441 ccgctacagg gcgcgtacta tggttgcttt gacgtatgcg gtgtgaaata ccgcacagat
     4501 gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg
     4561 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     4621 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
     4681 ccagtgagcg cttaacttcg tataaagtct cctatacgaa gttatcgtga ggaagagttc
     4741 ttgcagcgct taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     4801 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     4861 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg
     4921 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcta
     4981 gagtcgcggc cgctttactt gtacagctcg tccatgccgc cggtggagtg gcggccctcg
     5041 gagcgttcgt actgttccac cacggtgtag tcctcgttgt gggaggtgat gtccaacttg
     5101 cggtcgacgt tgtaggcgcc gggcatctgc acgggcttct tggccttgta ggtggtcttg
     5161 aagtccgcca ggtagcggcc gccgtccttc aggcgcaggg ccatcttaat gtcgcccttc
     5221 agcacgccgt cctcggggta caaccgctcg gtggacgctt cccagcccat tgtcttcttc
     5281 tgcattacgg ggccgtcagg agggaagttg gtgccgcgga gcttcacctt gtagatcagg
     5341 gtgccgtcct ccagggaggt gtcctgggtc acggtcacgg cgccgccgtc ctcgaagttc
     5401 atcacgcgct cccacttgaa gccctcgggg aaggactgct tatagtagtc ggggatgtcg
     5461 gcggggtgct tgatgaaggc cctggagccg tacatgaact gaggggacag gatgtcccag
     5521 gagaagggca gggggccacc cttggtcacc ttcagcttgg cggtctgggt gccctcgtag
     5581 gggcggccct cgccctcgcc ctcgatctcg aactcgtggc cgttcatgga gccctccatg
     5641 tgcaccttga accgcatgaa ctccttgatc actgcctcgc ccttgctcac catggtggcg
     5701 accggtggat ccgatccaga tccgcccgct tcgaagagca ggcgcagctc cccgacgctc
     5761 cggcgcggag cccgctccaa cccggggctg cagggccccg cgccccgtgg tcgcgccatg
     5821 cccatctgta ggaaaaagaa gaaggcatga acatggttag cagaggctct agagccgccg
     5881 gtcacacgcc agaagccgaa ccccgccctg ccccgtcccc cccgaaggca gccgtccccc
     5941 cgcggacagc cccgaggctg gagagggaga aggggacggc ggcgcggcga cgcacgaagg
     6001 ccctccccgc ccatttcctt cctgccggcg ccgcaccgct tcgccccgcg cccgctagag
     6061 ggggtgcggc ggcgcctccc agatttcggc tccgcacaga tttgggacaa aggaagtccc
     6121 tgcgccctct cgcacgatta ccataaaagg caatggctgc ggctcgccgc gcctcgacag
     6181 ccgccggcgc tccgggggcc gccgcgcccc tcccccgagc cctccccggc ccgaggcggc
     6241 cccgccccgc ccggcacccc cacctgccgc caccccccgc ccggcacggc gagccccgcg
     6301 ccacgccccg tacggagccc cgcacccgaa gccgggccgt gctcagcaac tcggggaggg
     6361 gggtgcaggg ggggttgcag cccgaccgac gcgcccacac cccctgctca cccccccacg
     6421 cacacacccc gcacgcagcc tttgttcccc tcgcagcccc ccccgcaccg cggggcaccg
     6481 cccccggccg cgctcccctc gcgcacactg cggagcgcac aaagccccgc gccgcgcccg
     6541 cagcgctcac agccgccggg cagcgcggag ccgcacgcgg cgctccccac gcacacacac
     6601 acgcacgcac cccccgagcc gctccccccg cacaaagggc cctcccggag cccctcaagg
     6661 ctttcacgca gccacagaaa agaaacaagc cgtcattaaa ccaagcgcta attacagccc
     6721 ggaggagaag ggccgtcccg cccgctcacc tgtgggagta acgcggtcag tcagagccgg
     6781 ggcgggcggc gcgaggcggc ggcggagcgg ggcacggggc gaaggcagcg cgcagcgact
     6841 cccgcccgcc gcgcgcttcg ctttttatag ggccgccgcc gccgccgcct cgccataaaa
     6901 ggaaactttc ggagcgcgcc gctctgattg gctgccgccg cacctctccg cctcgccccg
     6961 ccccgcccct cgccccgccc cgccccgcct ggcgcgcgcc cccccccccc gcccccatcg
     7021 ctgcacaaaa taattaaaaa ataaataaat acaaaattgg gggtggggag gggggggaga
     7081 tggggagagt gaagcagaac gtggggctca cctcgaccat ggtaatagcg atgactaata
     7141 cgtagatgta ctgccaagta ggaaagtccc ataaggtcat gtactgggca taatgccagg
     7201 cgggccattt accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat
     7261 gtactgccaa gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc
     7321 ctattggcgt tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg
     7381 gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac gcggaactta attaactagt
     7441 cgatgcctgc ttgccgaata tcatgataac ttcgtataat gtatgct
//
