LOCUS       sequence-260958-        6562 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector. It will generate the human TRAAK 
            channel full lenght G124I 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(G124I)
FEATURES             Location/Qualifiers
     source          1..6562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(1353..1927,1928..1930,1931..1939)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1520..1537)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1747..1766
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1940..1942,1943..1945,1946..2659)
                     /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(1940..1942,1943..1945,1946..2656)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1985..2006)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2246..2265)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2593..2614
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2782..2903
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2819..2838)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2873..2892
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2910..3365)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3047..3068)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3259..3278
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3392..3496
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3498..3855
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3498..3689
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3521..3541)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3706..3841
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3890..4684
                     /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             3890..4684
                     /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(3944..3963)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4554..4573
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4872..4891)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4916..4963
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5292..5880
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5781..5800
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        6055..6358
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6359..6562
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6513..6533
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 ggtttagtga accgtcagat ccgctagcgc taccggactc agatctcgag ccaccatgac
       61 taccgctcca caagaaccac ccgctcgccc actccaagct ggctccggtg ctggtcctgc
      121 tcctggacgt gctatgagat caacaacgct cctcgctctg ctggccctcg tattgctgta
      181 cctcgtgtct ggtgctctgg tattccgtgc tctcgaacaa ccacatgaac aacaggctca
      241 acgtgaactg ggagaagtcc gtgaaaaatt cctccgtgcc catccgtgtg tttccgatca
      301 agagttggga ttgctcatca aggaagtcgc cgacgctttg ggaggcggtg ccgaccctga
      361 gacaaactcc acctcaaact cctctcactc tgcctgggat ttgggatctg ccttcttctt
      421 ctctataacc atcatcacca ctatcggtta cggtaacgtg gcactccgta ccgacgccgg
      481 tagactgttc tgtattttct acgccctcgt tggtatccct ctcttcggaa tcttgttggc
      541 tggagtcgga gatcgcctcg gttcatcatt gaggcatggc atcggtcata ttgaggccat
      601 tttcttgaaa tggcacgtgc cacctgaatt ggttcgtgta ctgtccgcta tgctgttcct
      661 cctcatcggt tgcttgctct tcgttctcac ccccactttc gtattctgct acatggagga
      721 ctggtctaaa ctggaggcca tctacttcgt gatcgtgacc ctcactactg ttggattcgg
      781 agactacgtt gctggagccg atcctagaca ggattctcct gcctaccaac ctttggtgtg
      841 gttctggatt ctgttgggac tggcatactt cgctagtgtg ctcaccacca ttggcaactg
      901 gttgagagtg gtatcacgtc gtactcgtgc tgagatggga ggactcactg ctcaagctgc
      961 ttcatggacc ggaactgtga ctgctagagt cacccaacgt gctggaccgg ctgctccacc
     1021 acctgaaaaa gaacaacccc tgctgcctcc tccgccttgt cccgcccaac cgctgggacg
     1081 tcctcgttct ccttcacctc ccgaaaaggc acaaccacct tcacccccta ctgcttccgc
     1141 tttggactac ccttctgaaa acctggcttt catcgacgaa tcttcggaca ctcaatccga
     1201 gcgtggatgt ccattgccta gggctcctag gggccgtcgt agacccaacc ctccccgtaa
     1261 acctgttagg cctcgtggac ctggtagacc gagagacaaa ggagtccctg tttagtagtc
     1321 tgcagtcgac ggtaccgcgg gcccgggatc cgcccctctc cctccccccc ccctaacgtt
     1381 actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc
     1441 atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc
     1501 attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag
     1561 gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg
     1621 cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat
     1681 acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga
     1741 gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc
     1801 cattgtatgg gatctgatct ggggcctcgg tacacatgct ttacatgtgt ttagtcgagg
     1861 ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga
     1921 tgataatatg gccacaacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1981 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     2041 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2101 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     2161 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2221 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2281 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2341 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2401 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2461 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     2521 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     2581 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     2641 ggacgagctg tacaagtaaa gcggccgcga ctctagatca taatcagcca taccacattt
     2701 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     2761 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     2821 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2881 tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt
     2941 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     3001 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     3061 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     3121 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     3181 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     3241 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     3301 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     3361 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3421 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3481 ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg
     3541 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3601 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3661 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3721 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3781 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3841 ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     3901 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     3961 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4021 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
     4081 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4141 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4201 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4261 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4321 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4381 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct
     4441 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4501 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4561 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4621 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4681 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     4741 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4801 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg
     4861 gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca
     4921 ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg
     4981 tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc
     5041 gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc
     5101 aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat
     5161 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5221 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5281 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5341 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5401 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     5461 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5521 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5581 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5641 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5701 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5761 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5821 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5881 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5941 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta
     6001 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     6061 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     6121 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     6181 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     6241 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     6301 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     6361 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     6421 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     6481 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     6541 ggaggtctat ataagcagag ct
//
