LOCUS       sequence-485729-        7080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-GA 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 7080)
  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 7080)
  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-UbC-mStayGold
FEATURES             Location/Qualifiers
     source          1..7080
                     /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    1808..1855
                     /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)."
     promoter        1913..3124
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     2961..2980
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3171..3848
                     /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    3863..4087
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4138..4171
                     /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(4179..4195)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4209..4225)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4218..4240)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4438..4893
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4525..4544)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4735..4756
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4910..4929)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5029..5051
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5089..5107)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5175..5279
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5280..5348,5349..6140)
                     /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(5498..5517)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6311..6899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6800..6819
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7053..7070
                     /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 gcggactccg tcgtcaggat catccggatc
     1801 cggatccgaa gttcctattc cgaagttcct attctctaga aagtatagga acttcatgat
     1861 attcggcaag caggcatcga ctagttaatt aacccgtgtc ggctccagat ctggcctccg
     1921 cgccgggttt tggcgcctcc cgcgggcgcc cccctcctca cggcgagcgc tgccacgtca
     1981 gacgaagggc gcagcgagcg tcctgatcct tccgcccgga cgctcaggac agcggcccgc
     2041 tgctcataag actcggcctt agaaccccag tatcagcaga aggacatttt aggacgggac
     2101 ttgggtgact ctagggcact ggttttcttt ccagagagcg gaacaggcga ggaaaagtag
     2161 tcccttctcg gcgattctgc ggagggatct ccgtggggcg gtgaacgccg atgattatat
     2221 aaggacgcgc cgggtgtggc acagctagtt ccgtcgcagc cgggatttgg gtcgcggttc
     2281 ttgtttgtgg atcgctgtga tcgtcacttg gtgagtagcg ggctgctggg ctggccgggg
     2341 ctttcgtggc cgccgggccg ctcggtggga cggaagcgtg tggagagacc gccaagggct
     2401 gtagtctggg tccgcgagca aggttgccct gaactggggg ttggggggag cgcagcaaaa
     2461 tggcggctgt tcccgagtct tgaatggaag acgcttgtga ggcgggctgt gaggtcgttg
     2521 aaacaaggtg gggggcatgg tgggcggcaa gaacccaagg tcttgaggcc ttcgctaatg
     2581 cgggaaagct cttattcggg tgagatgggc tggggcacca tctggggacc ctgacgtgaa
     2641 gtttgtcact gactggagaa ctcggtttgt cgtctgttgc gggggcggca gttatggcgg
     2701 tgccgttggg cagtgcaccc gtacctttgg gagcgcgcgc cctcgtcgtg tcgtgacgtc
     2761 acccgttctg ttggcttata atgcagggtg gggccacctg ccggtaggtg tgcggtaggc
     2821 ttttctccgt cgcaggacgc agggttcggg cctagggtag gctctcctga atcgacaggc
     2881 gccggacctc tggtgagggg agggataagt gaggcgtcag tttctttggt cggttttatg
     2941 tacctatctt cttaagtagc tgaagctccg gttttgaact atgcgctcgg ggttggcgag
     3001 tgtgttttgt gaagtttttt aggcaccttt tgaaatgtaa tcatttgggt caatatgtaa
     3061 ttttcagtgt tagactagta aattgtccgc taaattctgg ccgtttttgg cttttttgtt
     3121 agacgaagct tgggcccggg aattaattca ccatgcgggg ttctgatccg atggtgtcta
     3181 caggcgagga gctgtttacc ggcgtggtgc ccttcaagtt ccagctgaag ggcaccatca
     3241 acggcaagag cttcaccgtg gaaggcgagg gcgagggcaa tagccacgag ggcagccaca
     3301 aaggcaagta cgtgtgcacc agcggcaaac tgccaatgtc ttgggccgcc ctgggaacta
     3361 gcttcggcta tggcatgaag tactacacca agtaccccag cggcctgaag aactggttcc
     3421 acgaggtgat gcccgagggc ttcacctacg acagacacat ccagtacaag ggcgacggca
     3481 gcatccacgc caagcaccag cacttcatga agaacggcac ctaccacaac atcgtggagt
     3541 tcaccggcca ggacttcaag gagaacagcc ccgtgctgac cagcgacatg gacgtgagcc
     3601 tgcccaacga ggtgcagcac atccccatag atgacggcgt ggagtgcaca gtgaccctgc
     3661 agtaccctct gctgagcgac gagagcaagt gcgtggaggc ctaccagaac accatcatca
     3721 agcccctgca caatcagcca gcccccgatg tgccattcca ctggatcaga aagcagtaca
     3781 cccagagcaa ggacgacacc gaggagaggg accacatcat ccagagcgag acactggagg
     3841 cccacctgta acaacgttgc acctgtgcct tctagttgcc agccatctgt tgtttgcccc
     3901 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3961 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     4021 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     4081 tctatggcgc tcgctcgctg caagaactct tcctcacgga tccgaagttc ctattccgaa
     4141 gttcctattc ttcaaatagt ataggaactt cagcgctcac tggccgtcgt tttacaaagg
     4201 gcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     4261 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     4321 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat
     4381 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     4441 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4501 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4561 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4621 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     4681 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4741 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     4801 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4861 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     4921 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     4981 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     5041 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     5101 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     5161 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5221 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5281 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5341 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5401 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5461 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5521 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5581 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5641 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5701 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5761 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5821 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5881 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5941 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6001 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6061 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6121 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6181 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6241 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6301 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6361 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6421 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6481 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6541 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6601 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6661 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6721 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6781 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6841 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6901 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     6961 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     7021 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
//
