LOCUS       sequence-485735-        7011 bp    DNA     circular SYN 10-SEP-2025
DEFINITION  Bxb1-GT donor plasmid for integrating a transcription unit for 
            constitutive expression of mStayGold with UbC promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7011)
  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 7011)
  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-UbC-mStayGold
FEATURES             Location/Qualifiers
     source          1..7011
                     /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)."
     promoter        1894..3105
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     2942..2961
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3152..3829
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ando et al., 2023)"
                     /label=mStayGold(J)
                     /note="includes multiple mutations plus an N-terminal 
                     iinker sequence"
                     /translation="MVSTGEELFTGVVPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTSDMDVSLPNEVQHIPIDDGVECTVTL
                     QYPLLSDESKCVEAYQNTIIKPLHNQPAPDVPFHWIRKQYTQSKDDTEERDHIIQSETL
                     EAHL"
     polyA_signal    3844..4068
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(4100..4132)
                     /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(4140..4157)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4140..4156)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4149..4171)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4369..4824
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4456..4475)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4666..4687
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4841..4860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4960..4982
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5020..5038)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5106..5210
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5211..5279,5280..6071)
                     /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(5429..5448)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6242..6830
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6731..6750
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6984..7001
                     /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 taacccgtgt cggctccaga tctggcctcc gcgccgggtt ttggcgcctc
     1921 ccgcgggcgc ccccctcctc acggcgagcg ctgccacgtc agacgaaggg cgcagcgagc
     1981 gtcctgatcc ttccgcccgg acgctcagga cagcggcccg ctgctcataa gactcggcct
     2041 tagaacccca gtatcagcag aaggacattt taggacggga cttgggtgac tctagggcac
     2101 tggttttctt tccagagagc ggaacaggcg aggaaaagta gtcccttctc ggcgattctg
     2161 cggagggatc tccgtggggc ggtgaacgcc gatgattata taaggacgcg ccgggtgtgg
     2221 cacagctagt tccgtcgcag ccgggatttg ggtcgcggtt cttgtttgtg gatcgctgtg
     2281 atcgtcactt ggtgagtagc gggctgctgg gctggccggg gctttcgtgg ccgccgggcc
     2341 gctcggtggg acggaagcgt gtggagagac cgccaagggc tgtagtctgg gtccgcgagc
     2401 aaggttgccc tgaactgggg gttgggggga gcgcagcaaa atggcggctg ttcccgagtc
     2461 ttgaatggaa gacgcttgtg aggcgggctg tgaggtcgtt gaaacaaggt ggggggcatg
     2521 gtgggcggca agaacccaag gtcttgaggc cttcgctaat gcgggaaagc tcttattcgg
     2581 gtgagatggg ctggggcacc atctggggac cctgacgtga agtttgtcac tgactggaga
     2641 actcggtttg tcgtctgttg cgggggcggc agttatggcg gtgccgttgg gcagtgcacc
     2701 cgtacctttg ggagcgcgcg ccctcgtcgt gtcgtgacgt cacccgttct gttggcttat
     2761 aatgcagggt ggggccacct gccggtaggt gtgcggtagg cttttctccg tcgcaggacg
     2821 cagggttcgg gcctagggta ggctctcctg aatcgacagg cgccggacct ctggtgaggg
     2881 gagggataag tgaggcgtca gtttctttgg tcggttttat gtacctatct tcttaagtag
     2941 ctgaagctcc ggttttgaac tatgcgctcg gggttggcga gtgtgttttg tgaagttttt
     3001 taggcacctt ttgaaatgta atcatttggg tcaatatgta attttcagtg ttagactagt
     3061 aaattgtccg ctaaattctg gccgtttttg gcttttttgt tagacgaagc ttgggcccgg
     3121 gaattaattc accatgcggg gttctgatcc gatggtgtct acaggcgagg agctgtttac
     3181 cggcgtggtg cccttcaagt tccagctgaa gggcaccatc aacggcaaga gcttcaccgt
     3241 ggaaggcgag ggcgagggca atagccacga gggcagccac aaaggcaagt acgtgtgcac
     3301 cagcggcaaa ctgccaatgt cttgggccgc cctgggaact agcttcggct atggcatgaa
     3361 gtactacacc aagtacccca gcggcctgaa gaactggttc cacgaggtga tgcccgaggg
     3421 cttcacctac gacagacaca tccagtacaa gggcgacggc agcatccacg ccaagcacca
     3481 gcacttcatg aagaacggca cctaccacaa catcgtggag ttcaccggcc aggacttcaa
     3541 ggagaacagc cccgtgctga ccagcgacat ggacgtgagc ctgcccaacg aggtgcagca
     3601 catccccata gatgacggcg tggagtgcac agtgaccctg cagtaccctc tgctgagcga
     3661 cgagagcaag tgcgtggagg cctaccagaa caccatcatc aagcccctgc acaatcagcc
     3721 agcccccgat gtgccattcc actggatcag aaagcagtac acccagagca aggacgacac
     3781 cgaggagagg gaccacatca tccagagcga gacactggag gcccacctgt aacaacgttg
     3841 cacctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3901 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3961 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     4021 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggcg ctcgctcgct
     4081 gcaagaactc ttcctcacga taacttcgta taggagactt tatacgaagt taagcgctca
     4141 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     4201 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     4261 ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt
     4321 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     4381 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4441 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4501 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4561 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4621 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4681 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     4741 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4801 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     4861 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4921 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4981 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     5041 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     5101 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5161 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5221 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5281 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5341 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5401 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5461 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5521 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5581 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5641 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5701 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5761 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5821 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     5881 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     5941 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6001 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6061 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6121 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6181 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6241 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6301 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6361 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6421 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6481 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6541 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6601 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6661 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6721 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6781 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6841 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6901 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     6961 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g
//
