LOCUS       sequence-403632-        6167 bp    DNA     circular SYN 14-APR-2025
DEFINITION  A genetically encoded ratiometric optical indicator for chloride 
            ions, to detect changes in intracellular chloride concentration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6167)
  AUTHORS   Kuner T, Augustine GJ
  TITLE     A genetically encoded ratiometric indicator for chloride: capturing 
            chloride transients in cultured hippocampal neurons.
  JOURNAL   Neuron. 2000 Sep;27(3):447-59. doi: 10.1016/s0896-6273(00)00056-8.
  PUBMED    11055428
REFERENCE   2  (bases 1 to 6167)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Neuron"; date: "2000-09"; volume: "27"; issue: "3"; pages: "447-59"
COMMENT     Sequence Label: Clomeleon
FEATURES             Location/Qualifiers
     source          1..6167
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..25
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    142..163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(178..183,184..201,202..208)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    216..232
                     /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     221..243
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     240..256
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     240..256
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        285..664
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        665..868
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     819..839
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     865..889
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1099..1117
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1099..1116
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1130..1149
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             join(1208..1210,1211..1213,1214..1924)
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1949..1969
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1997..1999,2000..2002,2003..2713)
                     /codon_start=1
                     /product="enhanced yellow variant of GFP"
                     /label=Topaz YFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     regulatory      2738..2747
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2751..2885
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2788..2807)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2842..2861
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2954..3311
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2954..3145
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2977..2997)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3162..3297
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3331..3348)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3331..3347)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3340..3362)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3560..4015
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3647..3666)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3857..3878
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4032..4051)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4151..4173
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4211..4229)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4297..4401
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4402..4470,4471..5262)
                     /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(4620..4639)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5433..6021
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5922..5941
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
       61 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
      121 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
      181 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
      241 aggaaacagc tatgacatga ttacgaatta attcgagctc gcccgacatt gattattgac
      301 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      361 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      421 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      481 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      541 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      601 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      661 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      721 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      781 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      841 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg
      901 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg
      961 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag
     1021 agtctatagg cccaccccct tggcttcgtt agaacgcggc tacaattaat acataacctt
     1081 atgtatcata cacatacgat ttaggtgaca ctatagaata acatccactt tgcctttctc
     1141 tccacaggtg tccactccca ggtccaactg cacctcggtt ctatcgattg aattccccct
     1201 cggtaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     1261 gctggacggc gacgtaaacg gccacaggtt cagcgtgtcc ggcgagggcg agggcgatgc
     1321 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     1381 gcccaccctc gtgaccaccc tgacctgggg cgtgcagtgc ttcagccgct accccgacca
     1441 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgtac
     1501 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     1561 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     1621 ggggcacaag ctggagtaca actacatcag ccacaacgtc tatatcaccg ccgacaagca
     1681 gaagaacggc atcaaggccc acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     1741 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     1801 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     1861 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     1921 caagaagctc accggtagtg gcagtggtga gaatttgtac ttccaaggag gcggcagcgg
     1981 aggcactagt agtaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2041 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2101 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2161 cgtgccctgg cccaccctcg tgaccacctt cggctacggc gtgcagtgct tcgcccgcta
     2221 ccccgaccac atgcgccagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2281 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2341 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2401 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     2461 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     2521 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2581 gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa
     2641 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     2701 cgagctgtac aagaagcttt aattaggtga cagcttggcc gccatggccc aacttgttta
     2761 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     2821 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     2881 ggatcgggaa ttaattcggc gcagcaccat ggcctgaaat aacctctgaa agaggaactt
     2941 ggttaggtac cttctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     3001 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3061 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3121 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3181 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3241 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3301 gcttttgcaa aaagctgtta acagcttggc actggccgtc gttttacaac gtcgtgactg
     3361 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     3421 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     3481 cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3541 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3601 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3661 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3721 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     3781 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3841 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3901 ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3961 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     4021 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     4081 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4141 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4201 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     4261 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4321 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4381 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     4441 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     4501 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     4561 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     4621 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     4681 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     4741 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     4801 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     4861 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     4921 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     4981 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5041 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5101 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     5161 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     5221 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     5281 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5341 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5401 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5461 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5521 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     5581 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     5641 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     5701 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     5761 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     5821 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     5881 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5941 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6001 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6061 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     6121 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgac
//
