LOCUS       sequence-463591-        7489 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 7489)
  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 7489)
  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..7489
                     /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)."
     enhancer        1880..2183
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2185..2460
                     /label=chicken beta-actin promoter
     intron          2461..3477
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3401..3423
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      3604..3613
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3610..4305
                     /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(3743..3761)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4011..4030
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4431..4552
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4468..4487)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4522..4541
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(4578..4610)
                     /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(4618..4635)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4618..4634)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4627..4649)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4847..5302
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4934..4953)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5144..5165
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5319..5338)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5438..5460
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5498..5516)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5584..5688
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5689..5757,5758..6549)
                     /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(5907..5926)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6720..7308
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7209..7228
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7462..7479
                     /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 taagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     1921 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     1981 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     2041 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     2101 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     2161 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
     2221 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
     2281 cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg
     2341 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
     2401 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
     2461 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
     2521 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
     2581 ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa
     2641 agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc
     2701 gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc
     2761 tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg
     2821 ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     2881 tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac
     2941 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt
     3001 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
     3061 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc
     3121 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     3181 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
     3241 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
     3301 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg
     3361 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     3421 accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagatg
     3481 ggcatggcgc gaccacgggg cgcggggccc tgcagccccg ggttggagcg ggctccgcgc
     3541 cggagcgtcg gggagctgcg cctgctcttc gaagcgggcg gatctggatc ggatccaccg
     3601 gtcgccacca tggtgagcaa gggcgaggca gtgatcaagg agttcatgcg gttcaaggtg
     3661 cacatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     3721 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     3781 tcctgggaca tcctgtcccc tcagttcatg tacggctcca gggccttcat caagcacccc
     3841 gccgacatcc ccgactacta taagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg
     3901 atgaacttcg aggacggcgg cgccgtgacc gtgacccagg acacctccct ggaggacggc
     3961 accctgatct acaaggtgaa gctccgcggc accaacttcc ctcctgacgg ccccgtaatg
     4021 cagaagaaga caatgggctg ggaagcgtcc accgagcggt tgtaccccga ggacggcgtg
     4081 ctgaagggcg acattaagat ggccctgcgc ctgaaggacg gcggccgcta cctggcggac
     4141 ttcaagacca cctacaaggc caagaagccc gtgcagatgc ccggcgccta caacgtcgac
     4201 cgcaagttgg acatcacctc ccacaacgag gactacaccg tggtggaaca gtacgaacgc
     4261 tccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaaag cggccgcgac
     4321 tctagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     4381 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     4441 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4501 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taagcgctgc
     4561 aagaactctt cctcacgata acttcgtata ggagacttta tacgaagtta agcgctcact
     4621 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     4681 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     4741 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
     4801 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     4861 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4921 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4981 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5041 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     5101 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5161 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     5221 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5281 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     5341 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5401 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5461 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     5521 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     5581 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5641 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5701 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5761 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5821 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5881 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5941 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     6001 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     6061 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     6121 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     6181 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     6241 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     6301 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     6361 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     6421 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     6481 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6541 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6601 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6661 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6721 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6781 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6841 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6901 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6961 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     7021 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     7081 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     7141 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     7201 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     7261 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     7321 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     7381 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     7441 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaag
//
