LOCUS       sequence-485700-        8119 bp    DNA     circular SYN 10-SEP-2025
DEFINITION  Bxb1-GT donor plasmid for integrating a transcription unit for 
            constitutive expression of mStayGold with CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8119)
  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 8119)
  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-GT-CAG-mStayGold
FEATURES             Location/Qualifiers
     source          1..8119
                     /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    complement(1802..1835)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        1945..2248
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          2527..3543
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3467..3489
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3551..3570
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3631..4308
                     /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"
     misc_feature    4312..4899
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4364..4384)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4782..4793)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4952..5176
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(5208..5240)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(5248..5265)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5248..5264)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5257..5279)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5477..5932
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5564..5583)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5774..5795
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5949..5968)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6068..6090
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6128..6146)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6214..6318
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6319..6387,6388..7179)
                     /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(6537..6556)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7350..7938
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7839..7858
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8092..8109
                     /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 gcggtctccg tcgtcaggat catccggatc
     1801 cataacttcg tatagcatac attatacgaa gttatcatga tattcggcaa gcaggcatcg
     1861 actagttaat taagtactta ctactagtta ttaatagtaa tcaattacgg ggtcattagt
     1921 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     1981 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     2041 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     2101 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     2161 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     2221 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
     2281 ccccatctcc cccccctccc cccccccaat tttttttttt tttttttttt tatttttttg
     2341 gggagggggg gggggggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag
     2401 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
     2461 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
     2521 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
     2581 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
     2641 tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg
     2701 cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt
     2761 gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt
     2821 gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg
     2881 gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg
     2941 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc
     3001 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg
     3061 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc
     3121 ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc
     3181 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag
     3241 agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc
     3301 cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
     3361 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc
     3421 tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg
     3481 cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta
     3541 cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa aaatggccac
     3601 catgccacca tgatgcgggg ttctgatccg atggtgtcta caggcgagga gctgtttacc
     3661 ggcgtggtgc ccttcaagtt ccagctgaag ggcaccatca acggcaagag cttcaccgtg
     3721 gaaggcgagg gcgagggcaa tagccacgag ggcagccaca aaggcaagta cgtgtgcacc
     3781 agcggcaaac tgccaatgtc ttgggccgcc ctgggaacta gcttcggcta tggcatgaag
     3841 tactacacca agtaccccag cggcctgaag aactggttcc acgaggtgat gcccgagggc
     3901 ttcacctacg acagacacat ccagtacaag ggcgacggca gcatccacgc caagcaccag
     3961 cacttcatga agaacggcac ctaccacaac atcgtggagt tcaccggcca ggacttcaag
     4021 gagaacagcc ccgtgctgac cagcgacatg gacgtgagcc tgcccaacga ggtgcagcac
     4081 atccccatag atgacggcgt ggagtgcaca gtgaccctgc agtaccctct gctgagcgac
     4141 gagagcaagt gcgtggaggc ctaccagaac accatcatca agcccctgca caatcagcca
     4201 gcccccgatg tgccattcca ctggatcaga aagcagtaca cccagagcaa ggacgacacc
     4261 gaggagaggg accacatcat ccagagcgag acactggagg cccacctgta aaatcaacct
     4321 ctggattcaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     4381 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     4441 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     4501 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     4561 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     4621 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     4681 acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc gcctgtgttg
     4741 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     4801 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     4861 ctcagacgag tcggatctcc ctttgggccg cctccccgcc ctttgggccg cctccccgcg
     4921 ctagtgctgg ggatgcgtaa caacgttgca cctgtgcctt ctagttgcca gccatctgtt
     4981 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     5041 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     5101 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     5161 gcggtgggct ctatggcgct cgctcgctgc aagaactctt cctcacgata acttcgtata
     5221 ggagacttta tacgaagtta agcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     5281 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     5341 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     5401 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     5461 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     5521 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     5581 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5641 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     5701 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5761 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     5821 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     5881 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     5941 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     6001 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     6061 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     6121 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     6181 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     6241 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6301 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6361 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6421 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6481 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6541 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6601 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6661 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6721 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6781 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6841 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6901 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6961 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     7021 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     7081 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     7141 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7201 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7261 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7321 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7381 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     7441 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     7501 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7561 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7621 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7681 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7741 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7801 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7861 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7921 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7981 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     8041 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     8101 gtgagcgagg aagcggaag
//
