LOCUS       sequence-463590-        7737 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 7737)
  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 7737)
  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..7737
                     /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    1808..1855
                     /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)."
     enhancer        1900..2203
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2205..2477
                     /label=chicken beta-actin promoter
     intron          2478..3494
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3418..3440
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3502..3521
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3573..4268
                     /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"
     CDS             4287..4307
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4311..4331
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4335..4355
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4627..4748
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4664..4683)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4718..4737
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    4795..4828
                     /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(4836..4852)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4866..4882)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4875..4897)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5095..5550
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5182..5201)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5392..5413
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5567..5586)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5686..5708
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5746..5764)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5832..5936
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5937..6005,6006..6797)
                     /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(6155..6174)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6968..7556
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7457..7476
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7710..7727
                     /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 gcggactccg tcgtcaggat catccggatc
     1801 cggatccgaa gttcctattc cgaagttcct attctctaga aagtatagga acttcatgat
     1861 attcggcaag caggcatcga ctagttaatt aagtttccgc gttacataac ttacggtaaa
     1921 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     1981 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     2041 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     2101 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     2161 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
     2221 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
     2281 tttttaatta ttttgtgcag cgatgggggc gggggggggg gcgcgcgcca ggcggggcgg
     2341 ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg
     2401 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
     2461 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
     2521 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     2581 gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt
     2641 ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
     2701 gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc
     2761 ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg
     2821 aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag
     2881 gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc
     2941 tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg
     3001 ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg
     3061 gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg
     3121 gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt
     3181 aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg
     3241 ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga
     3301 aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc
     3361 agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt
     3421 tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct
     3481 tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca
     3541 aagaattccg atcgcgccac catcctggac ctatggagag cgacgagagc ggcctgcccg
     3601 ccatggagat cgagtgccgc atcaccggca ccctgaacgg cgtggagttc gagctggtgg
     3661 gcggcggaga gggcaccccc gagcagggcc gcatgaccaa caagatgaag agcaccaaag
     3721 gcgccctgac cttcagcccc tacctgctga gccacgtgat gggctacggc ttctaccact
     3781 tcggcaccta ccccagcggc tacgagaacc ccttcctgca cgccatcaac aacggcggct
     3841 acaccaacac ccgcatcgag aagtacgagg acggcggcgt gctgcacgtg agcttcagct
     3901 accgctacga ggccggccgc gtgatcggcg acttcaaggt gatgggcacc ggcttccccg
     3961 aggacagcgt gatcttcacc gacaagatca tccgcagcaa cgccaccgtg gagcacctgc
     4021 accccatggg cgataacgat ctggatggca gcttcacccg caccttcagc ctgcgcgacg
     4081 gcggctacta cagctccgtg gtggacagcc acatgcactt caagagcgcc atccacccca
     4141 gcatcctgca gaacgggggc cccatgttcg ccttccgccg cgtggaggag gatcacagca
     4201 acaccgagct gggcatcgtg gagtaccagc acgccttcaa gaccccggat gcagatgccg
     4261 gtgaagaact cagatctcga gctgatccaa aaaagaagag aaaggtagat ccaaaaaaga
     4321 agagaaaggt agatccaaaa aagaagagaa aggtaggatc caccggatct agccatggct
     4381 tcccgccgga ggtggaggag caggatgatg gcacgctgcc catgtcttgt gcccaggaga
     4441 gcgggatgga ccgtcaccct gcagcctgtg cttctgctag gatcaatgtg tagtgtacaa
     4501 gtaaagcggc cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt
     4561 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     4621 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4681 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4741 gtatcttaag gcggctgcaa gaactcttcc tcacggatcc gaagttccta ttccgaagtt
     4801 cctattcttc aaatagtata ggaacttcag cgctcactgg ccgtcgtttt acaaagggcg
     4861 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4921 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     4981 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt
     5041 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     5101 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     5161 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     5221 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     5281 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     5341 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     5401 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
     5461 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5521 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     5581 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5641 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5701 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     5761 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     5821 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5881 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5941 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     6001 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     6061 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     6121 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     6181 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     6241 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     6301 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     6361 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6421 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6481 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6541 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6601 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     6661 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6721 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6781 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6841 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6901 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6961 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     7021 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     7081 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     7141 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     7201 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     7261 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     7321 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     7381 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     7441 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     7501 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     7561 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     7621 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     7681 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaag
//
