LOCUS       sequence-485701-        6100 bp    DNA     circular SYN 10-SEP-2025
DEFINITION  Bxb1-GA donor plasmid for integrating a transcription unit for 
            constitutive expression of mStayGold with RSV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6100)
  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 6100)
  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-GA-RSV-mStayGold
FEATURES             Location/Qualifiers
     source          1..6100
                     /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        1902..2162
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     CDS             2191..2868
                     /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    2883..3107
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3199..3215)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3229..3245)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3238..3260)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3458..3913
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3545..3564)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3755..3776
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3930..3949)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4049..4071
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4109..4127)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4195..4299
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4300..4368,4369..5160)
                     /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(4518..4537)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5331..5919
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5820..5839
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6073..6090
                     /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 aagtacttac taatgtagtc ttatgaatac
     1921 tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca tgccttacaa ggagagaaaa
     1981 agcaccgtgc atgccgattg gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc
     2041 aacagacggg tctgacatgg attggacgaa ccactgaatt ccgcattgca gagatattgt
     2101 atttaagtgc ctagctcgat acaataaacg ccatttgacc attcaccaca ttggtgtgca
     2161 ccaatggcca ccatgcgggg ttctgatccg atggtgtcta caggcgagga gctgtttacc
     2221 ggcgtggtgc ccttcaagtt ccagctgaag ggcaccatca acggcaagag cttcaccgtg
     2281 gaaggcgagg gcgagggcaa tagccacgag ggcagccaca aaggcaagta cgtgtgcacc
     2341 agcggcaaac tgccaatgtc ttgggccgcc ctgggaacta gcttcggcta tggcatgaag
     2401 tactacacca agtaccccag cggcctgaag aactggttcc acgaggtgat gcccgagggc
     2461 ttcacctacg acagacacat ccagtacaag ggcgacggca gcatccacgc caagcaccag
     2521 cacttcatga agaacggcac ctaccacaac atcgtggagt tcaccggcca ggacttcaag
     2581 gagaacagcc ccgtgctgac cggcgacatg gacgtgagcc tgcccaacga ggtgcagcac
     2641 atccccatag atgacggcgt ggagtgcaca gtgaccctgc agtaccctct gctgagcgac
     2701 gagagcaagt gcgtggaggc ctaccagaac accatcatca agcccctgca caatcagcca
     2761 gcccccgatg tgccattcca ctggatcaga aagcagtaca cccagagcaa ggacgacacc
     2821 gaggagaggg accacatcat ccagagcgag acactggagg cccacctgta acaacgttgc
     2881 acctgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2941 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3001 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3061 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggcgc tcgctcgctg
     3121 caagaactct tcctcacgga tccgaagttc ctattccgaa gttcctattc ttcaaatagt
     3181 ataggaactt cagcgctcac tggccgtcgt tttacaaagg gcgaattcac tggccgtcgt
     3241 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     3301 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     3361 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     3421 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     3481 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     3541 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     3601 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3661 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3721 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3781 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     3841 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     3901 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3961 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4021 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4081 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     4141 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     4201 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4261 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     4321 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     4381 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     4441 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     4501 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     4561 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     4621 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     4681 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     4741 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     4801 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     4861 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     4921 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4981 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5041 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5101 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5161 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     5221 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     5281 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     5341 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     5401 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     5461 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     5521 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     5581 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5641 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5701 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     5761 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     5821 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     5881 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5941 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6001 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     6061 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
//
