LOCUS       sequence-260970-        6562 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector. It will generate the human TRAAK 
            channel full lenght W262S mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6562)
  AUTHORS   Lolicato M, Riegelhaupt PM, Arrigoni C, Clark KA, Minor DL Jr
  TITLE     Transmembrane helix straightening and buckling underlies activation 
            of mechanosensitive and thermosensitive K(2P) channels.
  JOURNAL   Neuron. 2014 Dec 17;84(6):1198-212. doi: 
            10.1016/j.neuron.2014.11.017. Epub 2014 Dec 11.
  PUBMED    25500157
REFERENCE   2  (bases 1 to 6562)
  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: 
            "10.1016/j.neuron.2014.11.017"; journalName: "Neuron"; date: 
            "2014-12-17- 17"; volume: "84"; issue: "6"; pages: "1198-212"
COMMENT     Sequence Label: pIRES:hTRAAK(W262S)
FEATURES             Location/Qualifiers
     source          1..6562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    join(1557..2131,2132..2134,2135..2143)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1724..1741)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1951..1970
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2144..2146,2147..2149,2150..2863)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2144..2146,2147..2149,2150..2860)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2189..2210)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2450..2469)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2797..2818
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2986..3107
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3023..3042)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3077..3096
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3114..3569)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3251..3272)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3463..3482
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3596..3700
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3702..4059
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3702..3893
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3725..3745)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3910..4045
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4094..4888
                     /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             4094..4888
                     /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(4148..4167)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4758..4777
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5076..5095)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5120..5167
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5496..6084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5985..6004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        6259..6562
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 actcagatct cgagccacca tgactaccgc tccacaagaa ccacccgctc gcccactcca
      301 agctggctcc ggtgctggtc ctgctcctgg acgtgctatg agatcaacaa cgctcctcgc
      361 tctgctggcc ctcgtattgc tgtacctcgt gtctggtgct ctggtattcc gtgctctcga
      421 acaaccacat gaacaacagg ctcaacgtga actgggagaa gtccgtgaaa aattcctccg
      481 tgcccatccg tgtgtttccg atcaagagtt gggattgctc atcaaggaag tcgccgacgc
      541 tttgggaggc ggtgccgacc ctgagacaaa ctccacctca aactcctctc actctgcctg
      601 ggatttggga tctgccttct tcttctctgg aaccatcatc accactatcg gttacggtaa
      661 cgtggcactc cgtaccgacg ccggtagact gttctgtatt ttctacgccc tcgttggtat
      721 ccctctcttc ggaatcttgt tggctggagt cggagatcgc ctcggttcat cattgaggca
      781 tggcatcggt catattgagg ccattttctt gaaatggcac gtgccacctg aattggttcg
      841 tgtactgtcc gctatgctgt tcctcctcat cggttgcttg ctcttcgttc tcacccccac
      901 tttcgtattc tgctacatgg aggactggtc taaactggag gccatctact tcgtgatcgt
      961 gaccctcact actgttggat tcggagacta cgttgctgga gccgatccta gacaggattc
     1021 tcctgcctac caacctttgg tgtcgttctg gattctgttg ggactggcat acttcgctag
     1081 tgtgctcacc accattggca actggttgag agtggtatca cgtcgtactc gtgctgagat
     1141 gggaggactc actgctcaag ctgcttcatg gaccggaact gtgactgcta gagtcaccca
     1201 acgtgctgga ccggctgctc caccacctga aaaagaacaa cccctgctgc ctcctccgcc
     1261 ttgtcccgcc caaccgctgg gacgtcctcg ttctccttca cctcccgaaa aggcacaacc
     1321 accttcaccc cctactgctt ccgctttgga ctacccttct gaaaacctgg ctttcatcga
     1381 cgaatcttcg gacactcaat ccgagcgtgg atgtccattg cctagggctc ctaggggccg
     1441 tcgtagaccc aaccctcccc gtaaacctgt taggcctcgt ggacctggta gaccgagaga
     1501 caaaggagtc cctgtttagt agtctgcagt cgacggtacc gcgggcccgg gatccgcccc
     1561 tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg
     1621 tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa
     1681 cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg
     1741 caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca
     1801 acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc
     1861 ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt
     1921 gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg
     1981 ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc tcggtacaca
     2041 tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac
     2101 gtggttttcc tttgaaaaac acgatgataa tatggccaca accatggtga gcaagggcga
     2161 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     2221 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     2281 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     2341 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     2401 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     2461 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     2521 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     2581 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     2641 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     2701 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     2761 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     2821 cgccgccggg atcactctcg gcatggacga gctgtacaag taaagcggcc gcgactctag
     2881 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     2941 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     3001 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3061 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt
     3121 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     3181 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     3241 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     3301 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     3361 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3421 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     3481 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     3541 cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg
     3601 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3661 accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag
     3721 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3781 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3841 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3901 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3961 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     4021 tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg
     4081 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     4141 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     4201 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     4261 cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     4321 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4381 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4441 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4501 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4561 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4621 gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     4681 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     4741 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4801 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4861 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     4921 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4981 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     5041 ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg
     5101 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg
     5161 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga
     5221 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg
     5281 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt
     5341 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5401 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5461 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5521 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5581 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5641 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5701 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5761 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5821 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5881 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5941 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6001 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     6061 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6121 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     6181 cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag
     6241 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     6301 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     6361 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     6421 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     6481 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     6541 attagtcatc gctattacca tg
//
