LOCUS       sequence-292435-        6387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector. It will generate the mouse TREK-1 
            channel full lenght G137I mutant fused to a N-terminal HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6387)
  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 6387)
  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(G137I)
FEATURES             Location/Qualifiers
     source          1..6387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        349..400
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     primer_bind     351..371
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             432..458
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    1692..1744
                     /label=MCS
                     /note="multiple cloning site"
     misc_feature    join(1746..2320,2321..2323,2324..2332)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1913..1930)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2140..2159
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2333..2335,2336..2338,2339..3052)
                     /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(2333..2335,2336..2338,2339..3049)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2378..2399)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2639..2658)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2986..3007
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3175..3296
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3212..3231)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3266..3285
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3303..3758)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3440..3461)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3652..3671
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3785..3889
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3891..4248
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3891..4082
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3914..3934)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4099..4234
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4283..5077
                     /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             4283..5077
                     /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(4337..4356)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4947..4966
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5265..5284)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5309..5356
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5685..6273
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6174..6193
                     /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 caaaatgtcg taacaactcc gccccattga cgcaaatggg
      361 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
      421 ccgctagcat gtacccatac gacgtgccag actacgctgc ggcccctgac ttgctggatc
      481 ccaagtctgc tgctcagaac tccaaaccga ggctctcatt ctcctcaaaa cccaccgtgc
      541 ttgcttcccg ggtggagagt gactcggcca ttaatgttat gaaatggaag acggtctcca
      601 cgattttcct ggtggtcgtc ctctacctga tcatcggagc cacggtgttc aaggcattgg
      661 agcagcctca ggagatttcc cagaggacca ccattgtgat ccagaagcag accttcatag
      721 cccagcatgc ctgcgtcaac tccaccgagc tggacgaact catccagcaa atagtggcag
      781 caataaacgc aggaattatc cccttaggaa acagctccaa tcaagttagt cactgggacc
      841 tcggaagctc tttcttcttt gctattactg ttatcacaac cataggattt ggaaacatct
      901 ccccacgaac tgaaggtgga aaaatattct gcatcatcta tgccttgctg ggaattcccc
      961 tctttggctt tctactggct ggggttggtg atcagctagg aactatattt ggaaaaggaa
     1021 ttgccaaagt ggaagacaca tttattaagt ggaatgttag tcagacgaag attcgtatca
     1081 tctccaccat catcttcatc ctgtttggct gtgtcctctt tgtggctctc cctgcggtca
     1141 tattcaagca catagaaggc tggagcgccc tggacgctat ctattttgtg gttatcactc
     1201 tgacgaccat tggatttgga gactacgtgg caggtggatc agacattgaa tatctggact
     1261 tctacaagcc tgtggtgtgg ttctggatcc tcgttgggct ggcctacttt gcagctgttc
     1321 tgagcatgat tggggactgg ctacgggtga tctctaagaa gacgaaggaa gaggtgggag
     1381 agttcagagc gcatgccgct gagtggacag ccaatgtcac ggccgagttc aaggaaacga
     1441 ggaggcggct gagcgtggag atctacgaca agttccagcg tgccacatcc gtgaagcgga
     1501 agctctccgc agagctggcg ggcaaccaca accaggaact gactccgtgt cgtcgcaccc
     1561 tgtctgtgaa ccacctgacc agcgagaggg aagtcctgcc tcccttgctg aaggctgaga
     1621 gcatctatct gaacggtctg acaccacact gtgctggtga ggacatagct gtcattgaga
     1681 acatgaagtg actcgagctc aagcttcgaa ttctgcagtc gacggtaccg cgggcccggg
     1741 atccgcccct ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc
     1801 cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg
     1861 gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc
     1921 aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga
     1981 agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg
     2041 tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag
     2101 tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc
     2161 aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct
     2221 cggtacacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac
     2281 cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa ccatggtgag
     2341 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     2401 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2461 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2521 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2581 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2641 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2701 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2761 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     2821 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     2881 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     2941 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3001 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aaagcggccg
     3061 cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     3121 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     3181 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3241 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc
     3301 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3361 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     3421 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     3481 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     3541 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     3601 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     3661 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     3721 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     3781 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3841 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     3901 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3961 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4021 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4081 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4141 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4201 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     4261 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4321 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4381 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4441 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     4501 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4561 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4621 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4681 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4741 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4801 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4861 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4921 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4981 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5041 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     5101 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     5161 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5221 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     5281 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     5341 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     5401 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     5461 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     5521 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5581 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5641 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5701 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     5761 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5821 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5881 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5941 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6001 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6061 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6121 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6181 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6241 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6301 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6361 gtggataacc gtattaccgc catgcat
//
