LOCUS       sequence-292434-        6555 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector. It will generate the mouse TREK-1 
            channel full lenght K271Q mutant fused to a N-terminal HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6555)
  AUTHORS   Lolicato M, Arrigoni C, Mori T, Sekioka Y, Bryant C, Clark KA, Minor
            DL Jr
  TITLE     K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity 
            filter binding site.
  JOURNAL   Nature. 2017 Jul 20;547(7663):364-368. doi: 10.1038/nature22988. 
            Epub 2017 Jul 10.
  PUBMED    28693035
REFERENCE   2  (bases 1 to 6555)
  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: 
            "10.1038/nature22988"; journalName: "Nature"; date: "2017-07-20- 
            20"; volume: "547"; issue: "7663"; pages: "364-368"
COMMENT     Sequence Label: pIRES:mTREK-1(K271Q)
FEATURES             Location/Qualifiers
     source          1..6555
                     /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             600..626
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    1860..1912
                     /label=MCS
                     /note="multiple cloning site"
     misc_feature    join(1914..2488,2489..2491,2492..2500)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2081..2098)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2308..2327
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2501..2503,2504..2506,2507..3220)
                     /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(2501..2503,2504..2506,2507..3217)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2546..2567)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2807..2826)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3154..3175
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3343..3464
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3380..3399)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3434..3453
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3471..3926)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3608..3629)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3820..3839
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3953..4057
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4059..4416
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4059..4250
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4082..4102)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4267..4402
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4451..5245
                     /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             4451..5245
                     /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(4505..4524)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5115..5134
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5433..5452)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5477..5524
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5853..6441
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6342..6361
                     /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 gctagcatgt
      601 acccatacga cgtgccagac tacgctgcgg cccctgactt gctggatccc aagtctgctg
      661 ctcagaactc caaaccgagg ctctcattct cctcaaaacc caccgtgctt gcttcccggg
      721 tggagagtga ctcggccatt aatgttatga aatggaagac ggtctccacg attttcctgg
      781 tggtcgtcct ctacctgatc atcggagcca cggtgttcaa ggcattggag cagcctcagg
      841 agatttccca gaggaccacc attgtgatcc agaagcagac cttcatagcc cagcatgcct
      901 gcgtcaactc caccgagctg gacgaactca tccagcaaat agtggcagca ataaacgcag
      961 gaattatccc cttaggaaac agctccaatc aagttagtca ctgggacctc ggaagctctt
     1021 tcttctttgc tggtactgtt atcacaacca taggatttgg aaacatctcc ccacgaactg
     1081 aaggtggaaa aatattctgc atcatctatg ccttgctggg aattcccctc tttggctttc
     1141 tactggctgg ggttggtgat cagctaggaa ctatatttgg aaaaggaatt gccaaagtgg
     1201 aagacacatt tattaagtgg aatgttagtc agacgaagat tcgtatcatc tccaccatca
     1261 tcttcatcct gtttggctgt gtcctctttg tggctctccc tgcggtcata ttcaagcaca
     1321 tagaaggctg gagcgccctg gacgctatct attttgtggt tatcactctg acgaccattg
     1381 gatttggaga ctacgtggca ggtggatcag acattgaata tctggacttc taccagcctg
     1441 tggtgtggtt ctggatcctc gttgggctgg cctactttgc agctgttctg agcatgattg
     1501 gggactggct acgggtgatc tctaagaaga cgaaggaaga ggtgggagag ttcagagcgc
     1561 atgccgctga gtggacagcc aatgtcacgg ccgagttcaa ggaaacgagg aggcggctga
     1621 gcgtggagat ctacgacaag ttccagcgtg ccacatccgt gaagcggaag ctctccgcag
     1681 agctggcggg caaccacaac caggaactga ctccgtgtcg tcgcaccctg tctgtgaacc
     1741 acctgaccag cgagagggaa gtcctgcctc ccttgctgaa ggctgagagc atctatctga
     1801 acggtctgac accacactgt gctggtgagg acatagctgt cattgagaac atgaagtgac
     1861 tcgagctcaa gcttcgaatt ctgcagtcga cggtaccgcg ggcccgggat ccgcccctct
     1921 ccctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt
     1981 gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct
     2041 ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa
     2101 ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg
     2161 tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc
     2221 caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg
     2281 agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg
     2341 aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtacacatgc
     2401 tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg
     2461 gttttccttt gaaaaacacg atgataatat ggccacaacc atggtgagca agggcgagga
     2521 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     2581 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     2641 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     2701 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     2761 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     2821 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     2881 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     2941 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     3001 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     3061 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     3121 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     3181 cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc
     3241 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     3301 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     3361 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     3421 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag
     3481 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     3541 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     3601 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     3661 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     3721 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     3781 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     3841 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     3901 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     3961 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4021 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     4081 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4141 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     4201 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     4261 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     4321 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     4381 tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg
     4441 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     4501 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     4561 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     4621 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     4681 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     4741 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     4801 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     4861 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     4921 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     4981 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     5041 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     5101 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     5161 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     5221 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     5281 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     5341 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     5401 gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa
     5461 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt
     5521 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     5581 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     5641 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac
     5701 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5761 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5821 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5881 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5941 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6001 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6061 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6121 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6181 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6241 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6301 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6361 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6421 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6481 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     6541 attaccgcca tgcat
//
