LOCUS       sequence-485724-        6361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-GA donor plasmid for integrating a transcription unit for 
            constitutive expression of mStayGold with PGK promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6361)
  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 6361)
  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-PGK-mStayGold
FEATURES             Location/Qualifiers
     source          1..6361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        49..559
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     478..496
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             589..1266
                     /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
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMDVSLPNEVQHIPIDDGVECTVTL
                     QYPLLSDESKCVEAYQNTIIKPLHNQPAPDVPFHWIRKQYTQSKDDTEERDHIIQSETL
                     EAHL"
     polyA_signal    1281..1505
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    1556..1589
                     /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(1597..1613)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1627..1643)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1636..1658)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1856..2311
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1943..1962)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2153..2174
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2328..2347)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2447..2469
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2507..2525)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2593..2697
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2698..2766,2767..3558)
                     /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(2916..2935)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3729..4317
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4218..4237
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4471..4488
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4605..4626
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4641..4646,4647..4664,4665..4671)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4679..4695
                     /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     4684..4706
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4703..4719
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4703..4719
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4751..4961
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        4773..4969
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4820..4955
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        5056..6234
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5287..6225
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     6182..6202
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      6242..6251
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    6254..6291
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    6306..6353
                     /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)."
ORIGIN
        1 cggcaagcag gcatcgacta gttaattaag tacttactat aaggatccgg ggttggggtt
       61 gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc
      121 cgggaaacgc agcggcgccg accctggatc tcgcacattc ttcacgtccg ttcgcagcgt
      181 cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg ctccgcccct
      241 aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg
      301 tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc gccgaccgcg
      361 atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg gccgggaagg
      421 ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg
      481 ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat
      541 caccgacctc tctccccaga aatggccacc atgcggggtt ctgatccgat ggtgtctaca
      601 ggcgaggagc tgtttaccgg cgtggtgccc ttcaagttcc agctgaaggg caccatcaac
      661 ggcaagagct tcaccgtgga aggcgagggc gagggcaata gccacgaggg cagccacaaa
      721 ggcaagtacg tgtgcaccag cggcaaactg ccaatgtctt gggccgccct gggaactagc
      781 ttcggctatg gcatgaagta ctacaccaag taccccagcg gcctgaagaa ctggttccac
      841 gaggtgatgc ccgagggctt cacctacgac agacacatcc agtacaaggg cgacggcagc
      901 atccacgcca agcaccagca cttcatgaag aacggcacct accacaacat cgtggagttc
      961 accggccagg acttcaagga gaacagcccc gtgctgaccg gcgacatgga cgtgagcctg
     1021 cccaacgagg tgcagcacat ccccatagat gacggcgtgg agtgcacagt gaccctgcag
     1081 taccctctgc tgagcgacga gagcaagtgc gtggaggcct accagaacac catcatcaag
     1141 cccctgcaca atcagccagc ccccgatgtg ccattccact ggatcagaaa gcagtacacc
     1201 cagagcaagg acgacaccga ggagagggac cacatcatcc agagcgagac actggaggcc
     1261 cacctgtaac aacgttgcac ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     1321 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     1381 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     1441 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     1501 tatggcgctc gctcgctgca agaactcttc ctcacggatc cgaagttcct attccgaagt
     1561 tcctattctt caaatagtat aggaacttca gcgctcactg gccgtcgttt tacaaagggc
     1621 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     1681 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     1741 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     1801 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     1861 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     1921 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     1981 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2041 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     2101 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2161 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     2221 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2281 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     2341 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     2401 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     2461 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     2521 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     2581 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2641 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     2701 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     2761 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     2821 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     2881 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     2941 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3001 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3061 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3121 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3181 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3241 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3301 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3361 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     3421 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3481 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3541 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3601 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3661 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3721 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3781 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3841 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     3901 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3961 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4021 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4081 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4141 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4201 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4261 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4321 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4381 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4441 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4501 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     4561 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     4621 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     4681 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcttaat
     4741 tccctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     4801 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     4861 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     4921 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctttgcaa
     4981 agatggataa agttttaaac agagaggaat ctttgcagct aatggacctt ctaggtcttg
     5041 aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc
     5101 cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg
     5161 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     5221 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     5281 acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct
     5341 tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg
     5401 gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct
     5461 tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc
     5521 gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg
     5581 acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt
     5641 tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg
     5701 aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg
     5761 cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg
     5821 gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg
     5881 agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg
     5941 aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg
     6001 tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag
     6061 gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct
     6121 tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt
     6181 ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgaggaatt
     6241 cgccaccatg gccggcttgt cgacgacggc ggactccgtc gtcaggatca tccggatccg
     6301 gatccgaagt tcctattccg aagttcctat tctctagaaa gtataggaac ttcatgatat
     6361 t
//
