LOCUS       sequence-485705-        7695 bp    DNA     circular SYN 10-SEP-2025
DEFINITION  Bxb1-GA donor plasmid for integrating a transcription unit for 
            constitutive expression of mStayGold with CpG-free promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7695)
  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 7695)
  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-CpG-free-mStayGold
FEATURES             Location/Qualifiers
     source          1..7695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(161..177)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(161..177)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(174..196)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    185..201
                     /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)."
     promoter        complement(join(209..215,216..233,234..239))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    254..275
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(392..409)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(563..1151)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(643..662)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2660..3451,3452..3520))
                     /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     3283..3302
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3521..3625)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3693..3711
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3749..3771)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3871..3890
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3907..4362)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4044..4065)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4256..4275
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4560..4582
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4575..4591
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4605..4621
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    4713..4937
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(4952..5629)
                     /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"
     primer_bind     complement(5683..5702)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          5685..5914
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     protein_bind    complement(6222..6269)
                     /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)."
     protein_bind    6284..6321
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      6324..6333
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6341..7519)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6350..7288
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(6373..6393)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(join(7606..7695,1..107))
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(join(7614..7695,1..129))
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      join(7620..7695,1..60)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat
       61 gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact
      121 tctgcctgct ggggagggaa ttaagcttgg cgtaatcatg gtcatagctg tttcctgtgt
      181 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
      241 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
      301 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
      361 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
      421 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
      481 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
      541 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
      601 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
      661 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
      721 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
      781 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
      841 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
      901 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
      961 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     1021 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     1081 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     1141 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     1201 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     1261 taaattaaaa atgaagtttt aaatcaatct aaagtactga tgaatcccct aatgattttt
     1321 atcaaaatca ttaagttaag gtagatacac atcttgtcat atgatcaaat ggtttcgcca
     1381 aaaatcaata atcagacaac aaaatgtgcg aactcgatat tttacacgac tctctttacc
     1441 aattctgccc cgaattacac ttaaaacgac tcaacagctt aacgttggct tgccacgcct
     1501 tacttgactg taaaactctc actcttaccg aacttggccg taacctgcca accaaagcga
     1561 gaacaaaaca taacatcaaa cgaatcgacc gattgttagg taatcgtcac ctccacaaag
     1621 agcgactcgc tgtataccgt tggcatgcta gctttatctg ttcgggcaat acgatgccca
     1681 ttgtacttgt tgactggtct gatatccgtg agcaaaaacg gcttatggta ttgcgagctt
     1741 cagtcgcact acacggtcgt tctgttactc tttatgagaa agcgttcccg ctttcagagc
     1801 aatgttcaaa gaaagctcat gaccaatttc tagccgacct tgcgagcatt ctaccgagta
     1861 acaccacacc gctcattgtc agtgatgctg gctttaaagt gccatggtat aaatccgttg
     1921 agaagctggg ttggtactgg ttaagtcgag taagaggaaa agtacaatat gcagacctag
     1981 gagcggaaaa ctggaaacct atcagcaact tacatgatat gtcatctagt cactcaaaga
     2041 ctttaggcta taagaggctg actaaaagca atccaatctc atgccaaatt ctattgtata
     2101 aatctcgctc taaaggccga aaaaatcagc gctcgacacg gactcattgt caccacccgt
     2161 cacctaaaat ctactcagcg tcggcaaagg agccatggat tctagcaact aacttacctg
     2221 ttgaaattcg aacacccaaa caacttgtta atatctattc gaagcgaatg cagattgaag
     2281 aaaccttccg agacttgaaa agtcctgcct acggactagg cctacgccat agccgaacga
     2341 gcagctcaga gcgttttgat atcatgctgc taatcgccct gatgcttcaa ctaacatgtt
     2401 ggcttgcggg cgttcatgct cagaaacaag gttgggacaa gcacttccag gctaacacag
     2461 tcagaaatcg aaacgtactc tcaacagttc gcttaggcat ggaagttttg cggcattctg
     2521 gctacacaat aacaagggaa gacttactcg tggctgcaac cctactagct caaaatttat
     2581 tcacacatgg ttacgctttg gggaaattat gaggggatct ctcagtctaa agtatatatg
     2641 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     2701 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     2761 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     2821 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     2881 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     2941 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3001 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3061 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3121 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     3181 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     3241 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     3301 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     3361 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     3421 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     3481 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     3541 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     3601 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     3661 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     3721 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     3781 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     3841 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     3901 accataaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     3961 gctcattttt taaccaatag accgaaatcg gcaaaatccc ttataaatca aaagaatagc
     4021 ccgagataga gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     4081 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     4141 cacccaaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     4201 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     4261 agaaagcgaa aggagcgggc gctaaggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     4321 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtactatggt tgctttgacg
     4381 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc
     4441 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     4501 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     4561 ccagtcacga cgttgtaaaa cgacggccag tgaattcgcc ctttgtaaaa cgacggccag
     4621 tgagcgctga agttcctata ctatttgaag aataggaact tcggaatagg aacttcggat
     4681 ccgtgaggaa gagttcttgc agcgagcgag cgccatagag cccaccgcat ccccagcatg
     4741 cctgctattg tcttcccaat cctccccctt gctgtcctgc cccaccccac cccccagaat
     4801 agaatgacac ctactcagac aatgcgatgc aatttcctca ttttattagg aaaggacagt
     4861 gggagtggca ccttccaggg tcaaggaagg cacgggggag gggcaaacaa cagatggctg
     4921 gcaactagaa ggcacaggtg caacgttgtt acaggtgggc ctccagtgtc tcgctctgga
     4981 tgatgtggtc cctctcctcg gtgtcgtcct tgctctgggt gtactgcttt ctgatccagt
     5041 ggaatggcac atcgggggct ggctgattgt gcaggggctt gatgatggtg ttctggtagg
     5101 cctccacgca cttgctctcg tcgctcagca gagggtactg cagggtcact gtgcactcca
     5161 cgccgtcatc tatggggatg tgctgcacct cgttgggcag gctcacgtcc atgtcgccgg
     5221 tcagcacggg gctgttctcc ttgaagtcct ggccggtgaa ctccacgatg ttgtggtagg
     5281 tgccgttctt catgaagtgc tggtgcttgg cgtggatgct gccgtcgccc ttgtactgga
     5341 tgtgtctgtc gtaggtgaag ccctcgggca tcacctcgtg gaaccagttc ttcaggccgc
     5401 tggggtactt ggtgtagtac ttcatgccat agccgaagct agttcccagg gcggcccaag
     5461 acattggcag tttgccgctg gtgcacacgt acttgccttt gtggctgccc tcgtggctat
     5521 tgccctcgcc ctcgccttcc acggtgaagc tcttgccgtt gatggtgccc ttcagctgga
     5581 acttgaaggg caccacgccg gtaaacagct cctcgcctgt agacaccatc ggatcagaac
     5641 cccgcatggt ggccattcct gcagttggac ctgggagtgg acacctgtgg agagaaaggc
     5701 aaagtggatg tcattgtcac tcaagtgtat ggccagacct caagcctgcc acacctcaag
     5761 cttgacaaca aaaagattgt cttttctgac cagatggacc cagccaccct caaaggcatc
     5821 actgagggcc agttgaatat caaatcctcc tcatttttgg aaactgacaa tcttagtgca
     5881 gaagtcatgc cagcttttga gagggagtac tcaccccaac agctggccct ggcagacagt
     5941 gaattaattc aggccctgaa tgttcacaga gactactgca cttatatatg gttctccccc
     6001 accctgggga aaaaggtgga gccagtacac cacatcactt tcccagttta cccaagcccc
     6061 accttctcta ggcaccagtt caattgccca cccctccccc caacttctca gggactgtgg
     6121 gccatgtgct ctctgcccac tgaggggcac tcagccctca agcatgctct tctccagtaa
     6181 gtacttaatt aactagtcga tgcctgcttg ccgaatatca tgaagttcct atactttcta
     6241 gagaatagga acttcggaat aggaacttcg gatccggatc cggatgatcc tgacgacgga
     6301 gtccgccgtc gtcgacaagc cggccatggt ggcgaattcc tcacgacacc tgaaatggaa
     6361 gaaaaaaact ttgaaccact gtctgaggct tgagaatgaa ccaagatcca aactcaaaaa
     6421 gggcaaattc caaggagaat tacatcaagt gccaagctgg cctaacttca gtctccaccc
     6481 actcagtgtg gggaaactcc atcgcataaa acccctcccc ccaacctaaa gacgacgtac
     6541 tccaaaagct cgagaactaa tcgaggtgcc tggacggcgc ccggtactcc gtggagtcac
     6601 atgaagcgac ggctgaggac ggaaaggccc ttttcctttg tgtgggtgac tcacccgccc
     6661 gctctcccga gcgccgcgtc ctccattttg agctccctgc agcagggccg ggaagcggcc
     6721 atctttccgc tcacgcaact ggtgccgacc gggccagcct tgccgcccag ggcggggcga
     6781 tacacggcgg cgcgaggcca ggcaccagag caggccggcc agcttgagac tacccccgtc
     6841 cgattctcgg tggccgcgct cgcaggcccc gcctcgccga acatgtgcgc tgggacgcac
     6901 gggccccgtc gccgcccgcg gccccaaaaa ccgaaatacc agtgtgcaga tcttggcccg
     6961 catttacaag actatcttgc cagaaaaaaa gcgtcgcagc aggtcatcaa aaattttaaa
     7021 tggctagaga cttatcgaaa gcagcgagac aggcgcgaag gtgccaccag attcgcacgc
     7081 ggcggcccca gcgcccaggc caggcctcaa ctcaagcacg aggcgaaggg gctccttaag
     7141 cgcaaggcct cgaactctcc cacccacttc caacccgaag ctcgggatca agaatcacgt
     7201 actgcagcca ggtggaagta attcaaggca cgcaagggcc ataacccgta aagaggccag
     7261 gcccgcggga accacacacg gcacttacct gtgttctggc ggcaaacccg ttgcgaaaaa
     7321 gaacgttcac ggcgactact gcacttatat acggttctcc cccaccctcg ggaaaaaggc
     7381 ggagccagta cacgacatca ctttcccagt ttaccccgcg ccaccttctc taggcaccgg
     7441 ttcaattgcc gacccctccc cccaacttct cggggactgt gggcgatgtg cgctctgccc
     7501 actgacgggc accggagcca attcccactc ctttcaagac ctagaaggtc cattagctgc
     7561 aaagattcct ctctgtttaa aactttatcc atctttgcaa agctttttgc aaaagcctag
     7621 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg cggcctcggc
     7681 ctctgcataa ataaa
//
