LOCUS       sequence-463597-        7727 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-GT donor plasmid for integrating a transcription unit for 
            constitutive expression of nuclear-localised TurboGFP (3 copies).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7727)
  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 7727)
  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-GT_CAG_3xTurboGFP-NLS
FEATURES             Location/Qualifiers
     source          1..7727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..30)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(690..1481,1482..1550))
                     /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     1313..1332
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1551..1655)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1723..1741
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1779..1801)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1901..1920
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(1937..2392)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2074..2095)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2286..2305
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2590..2612
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2604..2621
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2605..2621
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2629..2661
                     /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)."
     polyA_signal    2689..2810
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2700..2719)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2754..2773
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3082..3102)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3106..3126)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3130..3150)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3169..3864)
                     /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(3916..3935)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          3943..4959
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(3997..4019)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     enhancer        5293..5596
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    5642..5675
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5684..5721
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      5724..5733
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5741..6919)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5750..6688
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(5773..5793)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(7006..7202)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(7014..7224)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7020..7155
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7256..7272)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7256..7272)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7269..7291)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7280..7296
                     /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(7304..7310,7311..7328,7329..7334))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7349..7370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7487..7504)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(7658..7727,1..519))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
       61 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
      121 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
      181 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
      241 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
      301 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
      361 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
      421 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
      481 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
      541 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
      601 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
      661 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
      721 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
      781 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
      841 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
      901 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
      961 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     1021 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     1081 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     1141 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     1201 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     1261 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     1321 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     1381 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     1441 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     1501 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     1561 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     1621 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     1681 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     1741 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     1801 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     1861 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
     1921 ctgagagtgc accataaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt
     1981 tgttaaatca gctcattttt taaccaatag accgaaatcg gcaaaatccc ttataaatca
     2041 aaagaatagc ccgagataga gttgagtgtt gttccagttt ggaacaagag tccactatta
     2101 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     2161 cgtgaaccat cacccaaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     2221 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     2281 aaggaaggga agaaagcgaa aggagcgggc gctaaggcgc tggcaagtgt agcggtcacg
     2341 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtactatggt
     2401 tgctttgacg tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     2461 ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     2521 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     2581 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgctta acttcgtata
     2641 aagtctccta tacgaagtta tcgtgaggaa gagttcttgc agccgcctta agatacattg
     2701 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     2761 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     2821 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     2881 aaaacctcta caaatgtggt atggctgatt atgatctaga gtcgcggccg ctttacttgt
     2941 acactacaca ttgatcctag cagaagcaca ggctgcaggg tgacggtcca tcccgctctc
     3001 ctgggcacaa gacatgggca gcgtgccatc atcctgctcc tccacctccg gcgggaagcc
     3061 atggctagat ccggtggatc ctacctttct cttctttttt ggatctacct ttctcttctt
     3121 ttttggatct acctttctct tcttttttgg atcagctcga gatctgagtt cttcaccggc
     3181 atctgcatcc ggggtcttga aggcgtgctg gtactccacg atgcccagct cggtgttgct
     3241 gtgatcctcc tccacgcggc ggaaggcgaa catggggccc ccgttctgca ggatgctggg
     3301 gtggatggcg ctcttgaagt gcatgtggct gtccaccacg gagctgtagt agccgccgtc
     3361 gcgcaggctg aaggtgcggg tgaagctgcc atccagatcg ttatcgccca tggggtgcag
     3421 gtgctccacg gtggcgttgc tgcggatgat cttgtcggtg aagatcacgc tgtcctcggg
     3481 gaagccggtg cccatcacct tgaagtcgcc gatcacgcgg ccggcctcgt agcggtagct
     3541 gaagctcacg tgcagcacgc cgccgtcctc gtacttctcg atgcgggtgt tggtgtagcc
     3601 gccgttgttg atggcgtgca ggaaggggtt ctcgtagccg ctggggtagg tgccgaagtg
     3661 gtagaagccg tagcccatca cgtggctcag caggtagggg ctgaaggtca gggcgccttt
     3721 ggtgctcttc atcttgttgg tcatgcggcc ctgctcgggg gtgccctctc cgccgcccac
     3781 cagctcgaac tccacgccgt tcagggtgcc ggtgatgcgg cactcgatct ccatggcggg
     3841 caggccgctc tcgtcgctct ccataggtcc aggatggtgg cgcgatcgga attctttgcc
     3901 aaaatgatga gacagcacaa taaccagcac gttgcccagg agctgtagga aaaagaagaa
     3961 ggcatgaaca tggttagcag aggctctaga gccgccggtc acacgccaga agccgaaccc
     4021 cgccctgccc cgtccccccc gaaggcagcc gtccccccgc ggacagcccc gaggctggag
     4081 agggagaagg ggacggcggc gcggcgacgc acgaaggccc tccccgccca tttccttcct
     4141 gccggcgccg caccgcttcg ccccgcgccc gctagagggg gtgcggcggc gcctcccaga
     4201 tttcggctcc gcacagattt gggacaaagg aagtccctgc gccctctcgc acgattacca
     4261 taaaaggcaa tggctgcggc tcgccgcgcc tcgacagccg ccggcgctcc gggggccgcc
     4321 gcgcccctcc cccgagccct ccccggcccg aggcggcccc gccccgcccg gcacccccac
     4381 ctgccgccac cccccgcccg gcacggcgag ccccgcgcca cgccccgtac ggagccccgc
     4441 acccgaagcc gggccgtgct cagcaactcg gggagggggg tgcagggggg gttgcagccc
     4501 gaccgacgcg cccacacccc ctgctcaccc ccccacgcac acaccccgca cgcagccttt
     4561 gttcccctcg cagccccccc cgcaccgcgg ggcaccgccc ccggccgcgc tcccctcgcg
     4621 cacactgcgg agcgcacaaa gccccgcgcc gcgcccgcag cgctcacagc cgccgggcag
     4681 cgcggagccg cacgcggcgc tccccacgca cacacacacg cacgcacccc ccgagccgct
     4741 ccccccgcac aaagggccct cccggagccc ctcaaggctt tcacgcagcc acagaaaaga
     4801 aacaagccgt cattaaacca agcgctaatt acagcccgga ggagaagggc cgtcccgccc
     4861 gctcacctgt gggagtaacg cggtcagtca gagccggggc gggcggcgcg aggcggcggc
     4921 ggagcggggc acggggcgaa ggcagcgcgc agcgactccc gcccgccgcg cgcttcgctt
     4981 tttatagggc cgccgccgcc gccgcctcgc cataaaagga aactttcgga gcgcgccgct
     5041 ctgattggct gccgccgcac ctctccgcct cgccccgccc cgcccctcgc cccgccccgc
     5101 cccgcccgcc gccccccccc cccccccccc cccccccccc cccccccccc ccccccgccc
     5161 cgcctggcgc gcgccccccc cccccgcccc catcgctgca caaaataatt aaaaaataaa
     5221 taaatacaaa attgggggtg gggagggggg ggagatgggg agagtgaagc agaacgtggg
     5281 gctcacctcg accatggtaa tagcgatgac taatacgtag atgtactgcc aagtaggaaa
     5341 gtcccataag gtcatgtact gggcataatg ccaggcgggc catttaccgt cattgacgtc
     5401 aatagggggc gtacttggca tatgatacac ttgatgtact gccaagtggg cagtttaccg
     5461 taaatactcc acccattgac gtcaatggaa agtccctatt ggcgttacta tgggaacata
     5521 cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg ggccatttac
     5581 cgtaagttat gtaacgcgga aacttaatta actagtcgat gcctgcttgc cgaatatcat
     5641 gataacttcg tataatgtat gctatacgaa gttatggatc cggatgatcc tgacgacgga
     5701 gaccgccgtc gtcgacaagc cggccatggt ggcgaattcc tcacgacacc tgaaatggaa
     5761 gaaaaaaact ttgaaccact gtctgaggct tgagaatgaa ccaagatcca aactcaaaaa
     5821 gggcaaattc caaggagaat tacatcaagt gccaagctgg cctaacttca gtctccaccc
     5881 actcagtgtg gggaaactcc atcgcataaa acccctcccc ccaacctaaa gacgacgtac
     5941 tccaaaagct cgagaactaa tcgaggtgcc tggacggcgc ccggtactcc gtggagtcac
     6001 atgaagcgac ggctgaggac ggaaaggccc ttttcctttg tgtgggtgac tcacccgccc
     6061 gctctcccga gcgccgcgtc ctccattttg agctccctgc agcagggccg ggaagcggcc
     6121 atctttccgc tcacgcaact ggtgccgacc gggccagcct tgccgcccag ggcggggcga
     6181 tacacggcgg cgcgaggcca ggcaccagag caggccggcc agcttgagac tacccccgtc
     6241 cgattctcgg tggccgcgct cgcaggcccc gcctcgccga acatgtgcgc tgggacgcac
     6301 gggccccgtc gccgcccgcg gccccaaaaa ccgaaatacc agtgtgcaga tcttggcccg
     6361 catttacaag actatcttgc cagaaaaaaa gcgtcgcagc aggtcatcaa aaattttaaa
     6421 tggctagaga cttatcgaaa gcagcgagac aggcgcgaag gtgccaccag attcgcacgc
     6481 ggcggcccca gcgcccaggc caggcctcaa ctcaagcacg aggcgaaggg gctccttaag
     6541 cgcaaggcct cgaactctcc cacccacttc caacccgaag ctcgggatca agaatcacgt
     6601 actgcagcca ggtggaagta attcaaggca cgcaagggcc ataacccgta aagaggccag
     6661 gcccgcggga accacacacg gcacttacct gtgttctggc ggcaaacccg ttgcgaaaaa
     6721 gaacgttcac ggcgactact gcacttatat acggttctcc cccaccctcg ggaaaaaggc
     6781 ggagccagta cacgacatca ctttcccagt ttaccccgcg ccaccttctc taggcaccgg
     6841 ttcaattgcc gacccctccc cccaacttct cggggactgt gggcgatgtg cgctctgccc
     6901 actgacgggc accggagcca attcccactc ctttcaagac ctagaaggtc cattagctgc
     6961 aaagattcct ctctgtttaa aactttatcc atctttgcaa agctttttgc aaaagcctag
     7021 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg cggcctcggc
     7081 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     7141 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg
     7201 catgctttgc atacttctgc ctgctgggga gggaattaag cttggcgtaa tcatggtcat
     7261 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     7321 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     7381 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     7441 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     7501 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     7561 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     7621 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     7681 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccg
//
