LOCUS       sequence-485703-        6050 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-GA donor plasmid for integrating a transcription unit for 
            constitutive expression of mStayGold with SV40 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6050)
  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 6050)
  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-SV40-mStayGold
FEATURES             Location/Qualifiers
     source          1..6050
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     69..89
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      129..138
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    141..178
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    193..240
                     /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        287..497
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      356..491
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             526..1203
                     /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    1218..1442
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    1493..1526
                     /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(1534..1550)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1564..1580)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1573..1595)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1793..2248
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1880..1899)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2090..2111
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2265..2284)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2384..2406
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2444..2462)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2530..2634
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2635..2703,2704..3495)
                     /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(2853..2872)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3666..4254
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4155..4174
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4408..4425
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4542..4563
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4578..4583,4584..4601,4602..4608)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4616..4632
                     /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     4621..4643
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4640..4656
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4640..4656
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4688..4898
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        4710..4906
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4757..4892
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        join(4993..6050,1..121)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          join(5224..6050,1..112)
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
ORIGIN
        1 gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg
       61 gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg
      121 aggaattcgc caccatggcc ggcttgtcga cgacggcgga ctccgtcgtc aggatcatcc
      181 ggatccggat ccgaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc
      241 atgatattcg gcaagcaggc atcgactagt taattaagta cttactgcag gcagaagtat
      301 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
      361 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
      421 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
      481 ttttggaggc ctaggctaat ggccaccatg cggggttctg atccgatggt gtctacaggc
      541 gaggagctgt ttaccggcgt ggtgcccttc aagttccagc tgaagggcac catcaacggc
      601 aagagcttca ccgtggaagg cgagggcgag ggcaatagcc acgagggcag ccacaaaggc
      661 aagtacgtgt gcaccagcgg caaactgcca atgtcttggg ccgccctggg aactagcttc
      721 ggctatggca tgaagtacta caccaagtac cccagcggcc tgaagaactg gttccacgag
      781 gtgatgcccg agggcttcac ctacgacaga cacatccagt acaagggcga cggcagcatc
      841 cacgccaagc accagcactt catgaagaac ggcacctacc acaacatcgt ggagttcacc
      901 ggccaggact tcaaggagaa cagccccgtg ctgaccagcg acatggacgt gagcctgccc
      961 aacgaggtgc agcacatccc catagatgac ggcgtggagt gcacagtgac cctgcagtac
     1021 cctctgctga gcgacgagag caagtgcgtg gaggcctacc agaacaccat catcaagccc
     1081 ctgcacaatc agccagcccc cgatgtgcca ttccactgga tcagaaagca gtacacccag
     1141 agcaaggacg acaccgagga gagggaccac atcatccaga gcgagacact ggaggcccac
     1201 ctgtaacaac gttgcacctg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     1261 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     1321 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     1381 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     1441 ggcgctcgct cgctgcaaga actcttcctc acggatccga agttcctatt ccgaagttcc
     1501 tattcttcaa atagtatagg aacttcagcg ctcactggcc gtcgttttac aaagggcgaa
     1561 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     1621 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     1681 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
     1741 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     1801 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     1861 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     1921 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     1981 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     2041 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2101 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     2161 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2221 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     2281 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     2341 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     2401 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     2461 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     2521 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2581 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     2641 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     2701 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     2761 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     2821 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     2881 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     2941 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     3001 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     3061 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     3121 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     3181 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     3241 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     3301 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     3361 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     3421 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     3481 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     3541 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3601 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3661 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3721 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3781 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     3841 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3901 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3961 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4021 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4081 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4141 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4201 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4261 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4321 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     4381 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     4441 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     4501 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     4561 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     4621 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcttaattcc
     4681 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     4741 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     4801 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     4861 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tttgcaaaga
     4921 tggataaagt tttaaacaga gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa
     4981 ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
     5041 gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta
     5101 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
     5161 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
     5221 caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc
     5281 gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt
     5341 ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga
     5401 gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc
     5461 tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg
     5521 ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg
     5581 gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg
     5641 cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct
     5701 gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc
     5761 cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc
     5821 tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa
     5881 agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc
     5941 aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg
     6001 ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag
//
