LOCUS       sequence-103771-        6328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent bile acid sensor (BAS) targeted to nucleus that allows 
            spatiotemporal monitoring of bile acid transport in single living 
            cells using FRET.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6328)
  AUTHORS   van der Velden LM, Golynskiy MV, Bijsmans IT, van Mil SW, Klomp LW, 
            Merkx M, van de Graaf SF
  TITLE     Monitoring bile acid transport in single living cells using a 
            genetically encoded Forster resonance energy transfer sensor.
  JOURNAL   Hepatology. 2013 Feb;57(2):740-52. doi: 10.1002/hep.26012. Epub 2013
            Jan 8.
  PUBMED    22899095
REFERENCE   2  (bases 1 to 6328)
  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"; doi: "10.1002/hep.26012";
            journalName: "Hepatology"; date: "2013-02"; volume: "57"; issue: 
            "2"; pages: "740-52"
COMMENT     Sequence Label: pCMV NucleoBAS
FEATURES             Location/Qualifiers
     source          1..6328
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(597..599,600..602,603..1115)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIE"
     primer_bind     complement(642..663)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(903..922)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2901..2921
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2940..2992
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    3116..3237
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3153..3172)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3207..3226
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3244..3699)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3381..3402)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3593..3612
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3726..3830
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3832..4189
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3832..4023
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3855..3875)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4040..4175
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4224..5018
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4224..5018
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4278..4297)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4888..4907
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5206..5225)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5250..5297
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5626..6214
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6115..6134
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcatgg
      601 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
      661 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
      721 agctgaccct gaagttcatc tgcaccaccg gtaagctgcc cgtgccctgg cccaccctcg
      781 tgaccaccct gacctggggc gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc
      841 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
      901 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
      961 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1021 tggagtacaa cgccatcagc gacaacgtct atatcaccgc cgacaagcag aagaacggca
     1081 tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     1141 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1201 tgagcacctt ctccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1261 tggagttcct gaccgccgcc gggatcacta gtgaactcac cccagatcaa cagactcttc
     1321 tacattttat tatggattca tataacaaac agaggatgcc tcaggaaata acaaataaaa
     1381 ttttaaaaga agaattcagt gcagaagaaa attttctcat tttgacggaa atggcaacca
     1441 atcatgtaca ggttcttgta gaattcacaa aaaagctacc aggatttcag actttggacc
     1501 atgaagacca gattgctttg ctgaaagggt ctgcggttga agctatgttc cttcgttcag
     1561 ctgagatttt caataagaaa cttccgtctg ggcattctga cctattggaa gaaagaattc
     1621 gaaatagtgg tatctctgat gaatatataa cacctatgtt tagtttttat aaaagtattg
     1681 gggaactgaa aatgactcaa gaggagtatg ctctgcttac agcaattgtt atcctgtctc
     1741 cagatagaca atacataaag gatagagagg cagtagagaa gcttcaggag ccacttcttg
     1801 atgtgctaca aaagttgtgt aagattcacc agcctgaaaa tcctcaacac tttgcctgtc
     1861 tcctgggtcg cctgactgaa ttacggacat tcaatcatca ccacgctgag atgctgatgt
     1921 catggagagt aaacgaccac aagtttaccc cacttctctg tgaaatctgg gacgtgcagg
     1981 gcgagctcgg cggatccggc ggaagcggcg gatccggcgg tagcggcgga tccggcggct
     2041 ccggcggatc cggcggcagc ggcggatccg gtggaagcgg tggatccggt ggtagcggtg
     2101 gatccggtgg aagcggtgga tccggtggta gcggtggatc cgggccgcgg ggtatggtga
     2161 gcaaaggcga agaactgttt actggtgttg tgccaattct ggtggaactg gatggcgatg
     2221 ttaacgggca caaattttcg gtttctggtg aaggtgaagg cgatgctaca tacggtaaac
     2281 tgacgctgaa atttatctgt accaccggca aactgccggt gccgtggccg actctggtta
     2341 caacgttcgg gtatggtctg atgtgctttg cccgctatcc agatcacatg aaacagcacg
     2401 acttcttcaa atcggctatg ccggaaggct atgtgcaaga acgtaccatc ttcttcaaag
     2461 atgatggcaa ctataaaacc cgcgctgaag tgaaatttga aggcgacacc ctggtcaatc
     2521 gcatcgaact gaaaggcatt gattttaaag aagatggcaa tattctgggc cataaactgg
     2581 aatacaatta taactcccat aatgtttaca ttatggcaga taaacagaaa aatggtatca
     2641 aagtgaattt caaaattcgc cacaatatcg aagatggttc ggtccagctg gcggaccatt
     2701 accagcagaa caccccgatc ggcgacggtc cggtgctgct gccagataac cattacctga
     2761 gttatttcag cgctctgtca aaagatccga atgaaaaacg tgatcacatg gtcctgctgg
     2821 aatttctgac ggcggcaggt attgcggccg ccgagaagca taaaattttg cacagactct
     2881 tgcaggacag cggcggtacc ccaaaaaaga agagaaaggt agaagacgcg ggtacctaac
     2941 tcgagctcaa gcttcgaatt ctgcagtcga cggtaccgcg ggcccgggat ccaccggatc
     3001 tagataactg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     3061 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     3121 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3181 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg
     3241 cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     3301 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     3361 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     3421 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     3481 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     3541 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     3601 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     3661 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa
     3721 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3781 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg
     3841 aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     3901 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     3961 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     4021 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     4081 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     4141 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag
     4201 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     4261 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     4321 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     4381 tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga
     4441 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     4501 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     4561 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     4621 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     4681 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     4741 ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     4801 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     4861 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     4921 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     4981 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     5041 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     5101 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     5161 ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga
     5221 gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg
     5281 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     5341 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     5401 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     5461 agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5521 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     5581 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5641 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5701 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5761 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5821 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5881 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5941 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6001 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6061 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6121 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6181 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6241 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     6301 tgtggataac cgtattaccg ccatgcat
//
