LOCUS       sequence-461675-        7550 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-GA donor plasmid for integrating a transcription unit for 
            constitutive expression of mStayGold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7550)
  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 7550)
  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_CAG-mStayGold
FEATURES             Location/Qualifiers
     source          1..7550
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    47..80
                     /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(88..104)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(118..134)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(127..149)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      347..802
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(434..453)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     644..665
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(819..838)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     938..960
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(998..1016)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1084..1188
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1189..1257,1258..2049)
                     /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(1407..1426)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2220..2808
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2709..2728
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2962..2979
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3096..3117
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3132..3137,3138..3155,3156..3162)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3170..3186
                     /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     3175..3197
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3194..3210
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3194..3210
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3242..3452
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        3264..3460
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3311..3446
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3547..4725
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3778..4716
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4673..4693
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4733..4742
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4745..4782
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    4797..4844
                     /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)."
     enhancer        4953..5256
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          5537..6553
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     6477..6499
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     6561..6580
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             6630..7307
                     /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    7322..7546
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 cgctcgctgc aagaactctt cctcacggat ccgaagttcc tattccgaag ttcctattct
       61 tcaaatagta taggaacttc agcgctcact ggccgtcgtt ttacaaaggg cgaattcact
      121 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
      181 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
      241 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
      301 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
      361 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
      421 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
      481 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
      541 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
      601 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
      661 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
      721 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
      781 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
      841 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
      901 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
      961 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     1021 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     1081 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1141 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     1201 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     1261 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     1321 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     1381 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     1441 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     1501 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     1561 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     1621 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     1681 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     1741 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     1801 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     1861 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     1921 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     1981 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     2041 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     2101 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     2161 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2221 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2281 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2341 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     2401 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2461 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2521 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2581 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2641 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2701 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2761 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2821 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     2881 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     2941 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     3001 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     3061 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     3121 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     3181 taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttaa ttccctcccc
     3241 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3301 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3361 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     3421 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctttgca aagatggata
     3481 aagttttaaa cagagaggaa tctttgcagc taatggacct tctaggtctt gaaaggagtg
     3541 ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     3601 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
     3661 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     3721 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta
     3781 agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct
     3841 tgaattactt ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt
     3901 gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag
     3961 gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc
     4021 gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt
     4081 ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt
     4141 ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc
     4201 ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg
     4261 gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg
     4321 gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa
     4381 tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc
     4441 tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac
     4501 ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat
     4561 gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg
     4621 atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct
     4681 cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgaggaat tcgccaccat
     4741 ggccggcttg tcgacgacgg cggactccgt cgtcaggatc atccggatcc ggatccgaag
     4801 ttcctattcc gaagttccta ttctctagaa agtataggaa cttcatgata ttcggcaagc
     4861 aggcatcgac tagttaatta agtacttact actagttatt aatagtaatc aattacgggg
     4921 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     4981 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     5041 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     5101 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     5161 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     5221 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
     5281 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
     5341 ttattttgtg cagcgatggg ggcggggggg gggggggggg ggggcccggg gggggggggg
     5401 gggggggggg gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg
     5461 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
     5521 aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc
     5581 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
     5641 ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt
     5701 tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg
     5761 ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc
     5821 cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga
     5881 ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg
     5941 ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct
     6001 gcaacccccc ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg
     6061 gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg
     6121 ggtgccgggc ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg
     6181 cccccggagc gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta
     6241 atcgtgcgag agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg
     6301 gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa
     6361 ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca
     6421 gcctcggggc tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt
     6481 cggcttctgg cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt
     6541 ctttttccta cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa
     6601 aaatggccac catgcggggt tctgatccga tggtgtctac aggcgaggag ctgtttaccg
     6661 gcgtggtgcc cttcaagttc cagctgaagg gcaccatcaa cggcaagagc ttcaccgtgg
     6721 aaggcgaggg cgagggcaat agccacgagg gcagccacaa aggcaagtac gtgtgcacca
     6781 gcggcaaact gccaatgtct tgggccgccc tgggaactag cttcggctat ggcatgaagt
     6841 actacaccaa gtaccccagc ggcctgaaga actggttcca cgaggtgatg cccgagggct
     6901 tcacctacga cagacacatc cagtacaagg gcgacggcag catccacgcc aagcaccagc
     6961 acttcatgaa gaacggcacc taccacaaca tcgtggagtt caccggccag gacttcaagg
     7021 agaacagccc cgtgctgacc agcgacatgg acgtgagcct gcccaacgag gtgcagcaca
     7081 tccccataga tgacggcgtg gagtgcacag tgaccctgca gtaccctctg ctgagcgacg
     7141 agagcaagtg cgtggaggcc taccagaaca ccatcatcaa gcccctgcac aatcagccag
     7201 cccccgatgt gccattccac tggatcagaa agcagtacac ccagagcaag gacgacaccg
     7261 aggagaggga ccacatcatc cagagcgaga cactggaggc ccacctgtaa caacgttgca
     7321 cctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     7381 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     7441 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     7501 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcgct
//
