LOCUS       sequence-204268-        7689 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression vector containing zebrafish Trpa1b with 
            C-terminal FLAG tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7689)
  AUTHORS   Lam PY, Mendu SK, Mills RW, Zheng B, Padilla H, Milan DJ, Desai BN, 
            Peterson RT
  TITLE     A high-conductance chemo-optogenetic system based on the vertebrate 
            channel Trpa1b.
  JOURNAL   Sci Rep. 2017 Sep 19;7(1):11839. doi: 10.1038/s41598-017-11791-z.
  PUBMED    28928472
REFERENCE   2  (bases 1 to 7689)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2017-09-19"; volume: "7(1):11839. doi"; pages: " 
            10.1038/s41598-017-11791-z"
COMMENT     Sequence Label: pCMV-Trpa1b-FLAG
FEATURES             Location/Qualifiers
     source          1..7689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      34..622
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     523..542
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        797..1100
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1101..1304
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1255..1275
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        1350..1368
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1455..1471)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1456..1472)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     regulatory      4816..4825
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4825..4890
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4920..4939)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4921..4939)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    5213..5334
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5250..5269)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5304..5323
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5341..5796)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5478..5499)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5690..5709
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5823..5927
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5929..6286
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5929..6120
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5952..5972)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6137..6272
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6321..7115
                     /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             6321..7115
                     /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(6375..6394)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6985..7004
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7303..7322)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7347..7394
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
       61 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
      121 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
      181 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
      241 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
      301 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
      361 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
      421 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
      481 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
      541 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
      601 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      661 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
      721 tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      781 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      841 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      901 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      961 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     1021 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atatacgtat
     1081 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     1141 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     1201 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     1261 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
     1321 agatccgcta gcgattacgc caagctcgaa attaaccctc actaaaggga acaaaagctg
     1381 gagctccacc gcggtggcgg ccgctctagc ccgggcggat cccccgggct gcaggaattc
     1441 gatatcaagc ttatcgatac cgtcgacctc gaggccacca tgcagtttgg aaaggagtta
     1501 gtgaggcgaa acagcttcta caaatgtgtg ataaacgagg acgaaacgga ggagtctgca
     1561 gatgtctttg agttggcttt caaaggaaag gcttctgcca tcgatcgtct gatccagaaa
     1621 ggtccagaac acctaagcct cagggatgag aatggagcca gtctgctgca ctatgcttca
     1681 gctggaggaa acctggatat cattcggctc attgtttcca ttgtgggccc tgaggtgctg
     1741 aatgtgcagg atgagcaggg ccgaactcct ctgcactggg ctgtggagca ggatcaacaa
     1801 cagagctgcg ctgtactgct ggatctgggt gctgacccca acattctcaa caatgccctg
     1861 atgggccctc tgcacctggc tgtcaccaaa caatacaatc atctagcaga ggtccttctg
     1921 tcttgtgaca aaaccaatag caatctggag ggggatctgg gaaacacccc tgtcatgctg
     1981 gcctgctcca ataacaactg ccaagctctc cagatcctga tcaagcgcgg tgccaagatg
     2041 tgtattcaga acaagctggg tcactatccc attcatactg ttgctttcgc aggtgccaaa
     2101 gaggccatgg agatggtatt gaaaattgga gaagaacttg gtgtatcatc tacattgcac
     2161 attaattatt tggataagtc caaaagcaca cctcttcatc tggctgtacg cggaggcaat
     2221 atcgaggtga tcaaactctg catcttgaaa ggagccaaag tagaacaaca tcagagtggc
     2281 aaatgcacgg ccctccactt tgcctgcagt cagggttctc tggaggctgt caaaatcatg
     2341 ctttcctctt acaacagaac agaagatatc gtcaacattc gggatggagc caatcggaca
     2401 cctttgcata gagcaacgct gtttgatcac gttgagttgg ctgaatatct tatttcaaag
     2461 ggagcagaaa ttgactctat tgactgtaag ggactgtctc cgcttctgct cgcctccagc
     2521 tgcagcgctt ggaaaacagt tgcatatctg ttgtccattg gtgcagattt taaaatcaaa
     2581 gaaaaaacag gtcgcaactt cctccacttt gtcattcttc aacccaaagg cctgaaaaat
     2641 cttcctgaga cggttttgca gagcaccgcc gtgaaggaga tgttgtctga tgaggatgtt
     2701 gaaggctgca ctcctctgca ctacgcctgc aagcttggca tccacgattc ggtcaaaaat
     2761 atgctgggtc tcaatatctg cttgggccaa aagtcacggg agaagaaatc agcgcttcac
     2821 ttcgctgcag aatatgggcg tataaataca tgccaccggc tgctggagac cctcacagac
     2881 tctaaaatgc tgaatgattg ggatgaaaag ggtctgacgc ccctccatct tgcttcaagg
     2941 gcaggacacg ctcaagtagt ggatctgctg cttaggaagg gagcactgtt tcaaagtgat
     3001 tacaagggct ggacctgttt acatcatgca gcagctgaag gatacacaca gacgatgaaa
     3061 atcctgctag cagccaatgt gaaactactg gatgaaaaaa atgaagatgg gaacacagcc
     3121 cttcatatag cagcacaagc tggacatgta agtgctgttt tgctgttatt ggacagagga
     3181 gctgaaatcg ctctcaacga tgctgacaac tcttttttgc atgaagctgt gcggaatgag
     3241 agaagagaag tagtaaacgc caccattgaa catgagcggt gtgatgagtc aatgacgtca
     3301 tttaaagcga aatcccgctg tgttgtgctg gacatcatag agttccttcc agaatctttt
     3361 cagcaccttc tggaccagtg tattacagaa tctgatcatg atgccaatag tcaggattat
     3421 catatcatgt acaatttcca atggcttcag gctcccatac agttgaagaa atatgctaaa
     3481 acagacaaaa caaaagcatt tcaaccactg gcagcattaa atgccatggt gcggtacaac
     3541 cgtctggagc tcctcattca ccccttgagc cgaaaatacc tggagatgaa gtggacggca
     3601 tatggaagca aagttcattt cctaaacttg gcgatatacc tgcttggatt gttgcccctc
     3661 acatacctca tcctcaatct gagacccagc caggacttct ccaaaggcaa tggcacatct
     3721 gtcatcatgg tgcccgtatc ttttagcgag caacaatatt taatctccgt gtgtataatc
     3781 atggtgatcg tcatgaatgt gtattcgatt tgcaaagagg tggtacagct ggctcagcag
     3841 cgagtaaatt acttcacaga tttctcaaac cctgcggact ggagtgcagc tattagcgct
     3901 ttggtttttg ttgtacccat gtgctgcagt gtggaggcca catggcaatg ggaagctgga
     3961 gcttatgcta ttctgacatc atggattggc tttctgcttt actttcagag atttgagcgg
     4021 attgggattt acgtggtgat gtttaatgga atcgtacgga ccctggtgtg catcatggtt
     4081 ctttttgtct tccttttgct ggctttcggc ttggctttct acgctctgat gctccacagg
     4141 ccggagttca gttccatcag cctagcgtta gcgcaaacgt ttgtgatgac agtaggagag
     4201 ctaaactacc aaagcacatt tcttaactcc tatgaagaag gccacatggc tttcccagcc
     4261 atcacttatt tagtttttgt gttttttgtt cttctcatgc ccattcttct catgaaccta
     4321 atgattggtt tggctgtagg agacattgca gaggtacaga gaaacgcaga gctgaaaagg
     4381 attgctatgc agatagatct tcacactgcc cttgaggaga aactacctta ctggttcctg
     4441 aagcgagtgg acaaaccctc taccgttgtc tatcccaata aatgcaagaa agtgcttctg
     4501 gaggccttgg tgcatggtga gattcagaac acagttcgaa ctcgtctaaa cctttgctca
     4561 cggaaggaag ggcttttgga gagagagctg cacaagcaga agaacaggtt gaaagaaatg
     4621 tcgtgcatgc tggaaaaaca gcataacctg ttgaagctga ttatccagaa gatggaaatt
     4681 acatctgaag ctgatgaata tgatggtcca cagaatcatg gagctctaaa acagcacacc
     4741 tcatccacct cacagaaatc aaagtgggtc ccgttactgc aagcaatcaa ggccaagaag
     4801 tcaggttgga ccggtgccac catggactat aaggaccacg acggagacta caaggatcat
     4861 gatattgatt acaaagacga tgacgataag tagggtacca ggtaagtgta cccaattcgc
     4921 cctatagtga gtcgtattac aattcactcg atcgcccttc ccaacagttg cgcagcctga
     4981 atggcgaatg gagatccaat ttttaagtgt ataatgtgtt aaactactga ttctaattgt
     5041 ttgtgtattt tagattcaca gtcccaaggc tcatttcagg cccctcagtc ctcacagtct
     5101 gttcatgatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     5161 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     5221 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5281 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt
     5341 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     5401 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     5461 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     5521 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     5581 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     5641 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     5701 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     5761 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg
     5821 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5881 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagaatcct gaggcggaaa
     5941 gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     6001 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     6061 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     6121 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     6181 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     6241 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga
     6301 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     6361 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     6421 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     6481 ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg
     6541 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     6601 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     6661 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     6721 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     6781 atcaggatga tctggacgaa gaacatcagg ggctcgcgcc agccgaactg ttcgccaggc
     6841 tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     6901 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     6961 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gaacttggcg
     7021 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     7081 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     7141 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     7201 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     7261 tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac
     7321 aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt
     7381 tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc
     7441 accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc
     7501 aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc
     7561 ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     7621 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     7681 ccactgagc
//
