LOCUS       sequence-463598-        7758 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-GA donor plasmid for integrating a transcription unit for 
            constitutive expression of nuclear-localised TurboGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7758)
  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 7758)
  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_CAG-TurboGFP
FEATURES             Location/Qualifiers
     source          1..7758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(152..740)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(232..251)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(911..1702,1703..1771))
                     /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     1534..1553
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1772..1876)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1944..1962
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2000..2022)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2122..2141
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2158..2613)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2295..2316)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2507..2526
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2811..2833
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2826..2842
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2856..2872
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2880..2913)
                     /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)"
     polyA_signal    2960..3081
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2971..2990)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3025..3044
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3353..3373)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3377..3397)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3401..3421)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3440..4135)
                     /codon_start=1
                     /product="improved green fluorescent protein from 
                     Pontellina plumata (Evdokimov et al., 2006)"
                     /label=TurboGFP
                     /note="mammalian codon-optimized"
                     /translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPEQGRMTNKM
                     KSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVL
                     HVSFSYRYEAGRVIGDFKVMGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNDLDGSFTR
                     TFSLRDGGYYSSVVDSHMHFKSAIHPSILQNGGPMFAFRRVEEDHSNTELGIVEYQHAF
                     KTPDADAGEE"
     primer_bind     complement(4187..4206)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          4214..5230
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(4268..4290)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     enhancer        5526..5829
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    complement(5874..5921)
                     /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    5936..5973
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      5976..5985
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5993..7171)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6002..6940
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(6025..6045)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(7258..7454)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(7266..7476)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7272..7407
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7508..7524)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7508..7524)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7521..7543)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7532..7548
                     /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(7556..7562,7563..7580,7581..7586))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7601..7622
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7739..7756)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
       61 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
      121 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
      181 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
      241 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
      301 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
      361 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
      421 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
      481 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
      541 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
      601 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
      661 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
      721 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
      781 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
      841 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
      901 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
      961 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     1021 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     1081 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     1141 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     1201 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     1261 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     1321 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     1381 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     1441 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     1501 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     1561 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     1621 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     1681 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     1741 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     1801 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     1861 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     1921 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     1981 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     2041 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     2101 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccataaaa
     2161 ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     2221 ttaaccaata gaccgaaatc ggcaaaatcc cttataaatc aaaagaatag cccgagatag
     2281 agttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     2341 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcacccaaat
     2401 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     2461 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2521 aaggagcggg cgctaaggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2581 ccgccgcgct taatgcgccg ctacagggcg cgtactatgg ttgctttgac gtatgcggtg
     2641 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag
     2701 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     2761 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     2821 acgttgtaaa acgacggcca gtgaattcgc cctttgtaaa acgacggcca gtgagcgctg
     2881 aagttcctat actatttgaa gaataggaac ttcggaatag gaacttcgga tccgtgagga
     2941 agagttcttg cagccgcctt aagatacatt gatgagtttg gacaaaccac aactagaatg
     3001 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     3061 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     3121 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat
     3181 tatgatctag agtcgcggcc gctttacttg tacactacac attgatccta gcagaagcac
     3241 aggctgcagg gtgacggtcc atcccgctct cctgggcaca agacatgggc agcgtgccat
     3301 catcctgctc ctccacctcc ggcgggaagc catggctaga tccggtggat cctacctttc
     3361 tcttcttttt tggatctacc tttctcttct tttttggatc tacctttctc ttcttttttg
     3421 gatcagctcg agatctgagt tcttcaccgg catctgcatc cggggtcttg aaggcgtgct
     3481 ggtactccac gatgcccagc tcggtgttgc tgtgatcctc ctccacgcgg cggaaggcga
     3541 acatggggcc cccgttctgc aggatgctgg ggtggatggc gctcttgaag tgcatgtggc
     3601 tgtccaccac ggagctgtag tagccgccgt cgcgcaggct gaaggtgcgg gtgaagctgc
     3661 catccagatc gttatcgccc atggggtgca ggtgctccac ggtggcgttg ctgcggatga
     3721 tcttgtcggt gaagatcacg ctgtcctcgg ggaagccggt gcccatcacc ttgaagtcgc
     3781 cgatcacgcg gccggcctcg tagcggtagc tgaagctcac gtgcagcacg ccgccgtcct
     3841 cgtacttctc gatgcgggtg ttggtgtagc cgccgttgtt gatggcgtgc aggaaggggt
     3901 tctcgtagcc gctggggtag gtgccgaagt ggtagaagcc gtagcccatc acgtggctca
     3961 gcaggtaggg gctgaaggtc agggcgcctt tggtgctctt catcttgttg gtcatgcggc
     4021 cctgctcggg ggtgccctct ccgccgccca ccagctcgaa ctccacgccg ttcagggtgc
     4081 cggtgatgcg gcactcgatc tccatggcgg gcaggccgct ctcgtcgctc tccataggtc
     4141 caggatggtg gcgcgatcgg aattctttgc caaaatgatg agacagcaca ataaccagca
     4201 cgttgcccag gagctgtagg aaaaagaaga aggcatgaac atggttagca gaggctctag
     4261 agccgccggt cacacgccag aagccgaacc ccgccctgcc ccgtcccccc cgaaggcagc
     4321 cgtccccccg cggacagccc cgaggctgga gagggagaag gggacggcgg cgcggcgacg
     4381 cacgaaggcc ctccccgccc atttccttcc tgccggcgcc gcaccgcttc gccccgcgcc
     4441 cgctagaggg ggtgcggcgg cgcctcccag atttcggctc cgcacagatt tgggacaaag
     4501 gaagtccctg cgccctctcg cacgattacc ataaaaggca atggctgcgg ctcgccgcgc
     4561 ctcgacagcc gccggcgctc cgggggccgc cgcgcccctc ccccgagccc tccccggccc
     4621 gaggcggccc cgccccgccc ggcaccccca cctgccgcca ccccccgccc ggcacggcga
     4681 gccccgcgcc acgccccgta cggagccccg cacccgaagc cgggccgtgc tcagcaactc
     4741 ggggaggggg gtgcaggggg ggttgcagcc cgaccgacgc gcccacaccc cctgctcacc
     4801 cccccacgca cacaccccgc acgcagcctt tgttcccctc gcagcccccc ccgcaccgcg
     4861 gggcaccgcc cccggccgcg ctcccctcgc gcacactgcg gagcgcacaa agccccgcgc
     4921 cgcgcccgca gcgctcacag ccgccgggca gcgcggagcc gcacgcggcg ctccccacgc
     4981 acacacacac gcacgcaccc cccgagccgc tccccccgca caaagggccc tcccggagcc
     5041 cctcaaggct ttcacgcagc cacagaaaag aaacaagccg tcattaaacc aagcgctaat
     5101 tacagcccgg aggagaaggg ccgtcccgcc cgctcacctg tgggagtaac gcggtcagtc
     5161 agagccgggg cgggcggcgc gaggcggcgg cggagcgggg cacggggcga aggcagcgcg
     5221 cagcgactcc cgcccgccgc gcgcttcgct ttttataggg ccgccgccgc cgccgcctcg
     5281 ccataaaagg aaactttcgg agcgcgccgc tctgattggc tgccgccgca cctctccgcc
     5341 tcgccccccc cccccccccc cccccccccc ccccccccct ccccccgccc cgccccgcct
     5401 ggcgcgcgcc ccccccccgc ccccatcgct gcacaaaata attaaaaaat aaataaatac
     5461 aaaattgggg gtggggaggg gggggagatg gggagagtga agcagaacgt ggggctcacc
     5521 tcgaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg aaagtcccat
     5581 aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg
     5641 ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac
     5701 tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac atacgtcatt
     5761 attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt
     5821 tatgtaacgc ggaaacttaa ttaactagtc gatgcctgct tgccgaatat catgaagttc
     5881 ctatactttc tagagaatag gaacttcgga ataggaactt cggatccgga tccggatgat
     5941 cctgacgacg gagtccgccg tcgtcgacaa gccggccatg gtggcgaatt cctcacgaca
     6001 cctgaaatgg aagaaaaaaa ctttgaacca ctgtctgagg cttgagaatg aaccaagatc
     6061 caaactcaaa aagggcaaat tccaaggaga attacatcaa gtgccaagct ggcctaactt
     6121 cagtctccac ccactcagtg tggggaaact ccatcgcata aaacccctcc ccccaaccta
     6181 aagacgacgt actccaaaag ctcgagaact aatcgaggtg cctggacggc gcccggtact
     6241 ccgtggagtc acatgaagcg acggctgagg acggaaaggc ccttttcctt tgtgtgggtg
     6301 actcacccgc ccgctctccc gagcgccgcg tcctccattt tgagctccct gcagcagggc
     6361 cgggaagcgg ccatctttcc gctcacgcaa ctggtgccga ccgggccagc cttgccgccc
     6421 agggcggggc gatacacggc ggcgcgaggc caggcaccag agcaggccgg ccagcttgag
     6481 actacccccg tccgattctc ggtggccgcg ctcgcaggcc ccgcctcgcc gaacatgtgc
     6541 gctgggacgc acgggccccg tcgccgcccg cggccccaaa aaccgaaata ccagtgtgca
     6601 gatcttggcc cgcatttaca agactatctt gccagaaaaa aagcgtcgca gcaggtcatc
     6661 aaaaatttta aatggctaga gacttatcga aagcagcgag acaggcgcga aggtgccacc
     6721 agattcgcac gcggcggccc cagcgcccag gccaggcctc aactcaagca cgaggcgaag
     6781 gggctcctta agcgcaaggc ctcgaactct cccacccact tccaacccga agctcgggat
     6841 caagaatcac gtactgcagc caggtggaag taattcaagg cacgcaaggg ccataacccg
     6901 taaagaggcc aggcccgcgg gaaccacaca cggcacttac ctgtgttctg gcggcaaacc
     6961 cgttgcgaaa aagaacgttc acggcgacta ctgcacttat atacggttct cccccaccct
     7021 cgggaaaaag gcggagccag tacacgacat cactttccca gtttaccccg cgccaccttc
     7081 tctaggcacc ggttcaattg ccgacccctc cccccaactt ctcggggact gtgggcgatg
     7141 tgcgctctgc ccactgacgg gcaccggagc caattcccac tcctttcaag acctagaagg
     7201 tccattagct gcaaagattc ctctctgttt aaaactttat ccatctttgc aaagcttttt
     7261 gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga
     7321 ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc
     7381 ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
     7441 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagggaatta agcttggcgt
     7501 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     7561 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     7621 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     7681 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     7741 cgctcactga ctcgctgc
//
