LOCUS       sequence-445191-        6555 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for generating mouse TREK-1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6555)
  AUTHORS   Deal PE, Lee H, Mondal A, Lolicato M, Mendonca PRF, Black H, Jang S,
            El-Hilali X, Bryant C, Isacoff EY, Renslo AR, Minor DL Jr
  TITLE     Development of covalent chemogenetic K(2P) channel activators.
  JOURNAL   Cell Chem Biol. 2024 Jul 18;31(7):1305-1323.e9. doi: 
            10.1016/j.chembiol.2024.06.006.
  PUBMED    39029456
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: 
            "10.1016/j.chembiol.2024.06"; journalName: "Cell Chem Biol"; date: 
            "2024-07-18- 18"; volume: "31"; issue: "7"; pages: "1305-1323.e9"
COMMENT     Sequence Label: pIRES2-eGFP:HA-mTREK-1(S131C/A286F)
FEATURES             Location/Qualifiers
     source          1..6555
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        764..1067
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1068..1271
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1222..1242
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1303..1329
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    2563..2615
                     /label=MCS
                     /note="multiple cloning site"
     misc_feature    join(2617..3191,3192..3194,3195..3203)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2784..2801)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3011..3030
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3204..3206,3207..3209,3210..3923)
                     /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(3204..3206,3207..3209,3210..3920)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3249..3270)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3510..3529)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3857..3878
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4046..4167
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4083..4102)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4137..4156
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4174..4629)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4311..4332)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4523..4542
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4656..4760
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4762..5119
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4762..4953
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4785..4805)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4970..5105
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5154..5948
                     /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             5154..5948
                     /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(5208..5227)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5818..5837
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6136..6155)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6180..6227
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
      721 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      781 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      841 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      901 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      961 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1021 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1081 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1141 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1201 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1261 taagcagagc tggtttagtg aaccgtcaga tccgctagca tgtacccata cgacgtgcca
     1321 gactacgctg cggcccctga cttgctggat cccaagtctg ctgctcagaa ctccaaaccg
     1381 aggctctcat tctcctcaaa acccaccgtg cttgcttccc gggtggagag tgactcggcc
     1441 attaatgtta tgaaatggaa gacggtctcc acgattttcc tggtggtcgt cctctacctg
     1501 atcatcggag ccacggtgtt caaggcattg gagcagcctc aggagatttc ccagaggacc
     1561 accattgtga tccagaagca gaccttcata gcccagcatg cctgcgtcaa ctccaccgag
     1621 ctggacgaac tcatccagca aatagtggca gcaataaacg caggaattat ccccttagga
     1681 aacagctcca atcaagttag tcactgggac ctcggatgct ctttcttctt tgctggtact
     1741 gttatcacaa ccataggatt tggaaacatc tccccacgaa ctgaaggtgg aaaaatattc
     1801 tgcatcatct atgccttgct gggaattccc ctctttggct ttctactggc tggggttggt
     1861 gatcagctag gaactatatt tggaaaagga attgccaaag tggaagacac atttattaag
     1921 tggaatgtta gtcagacgaa gattcgtatc atctccacca tcatcttcat cctgtttggc
     1981 tgtgtcctct ttgtggctct ccctgcggtc atattcaagc acatagaagg ctggagcgcc
     2041 ctggacgcta tctattttgt ggttatcact ctgacgacca ttggatttgg agactacgtg
     2101 gcaggtggat cagacattga atatctggac ttctacaagc ctgtggtgtg gttctggatc
     2161 ctcgttgggc tggcctactt tttcgctgtt ctgagcatga ttggggactg gctacgggtg
     2221 atctctaaga agacgaagga agaggtggga gagttcagag cgcatgccgc tgagtggaca
     2281 gccaatgtca cggccgagtt caaggaaacg aggaggcggc tgagcgtgga gatctacgac
     2341 aagttccagc gtgccacatc cgtgaagcgg aagctctccg cagagctggc gggcaaccac
     2401 aaccaggaac tgactccgtg tcgtcgcacc ctgtctgtga accacctgac cagcgagagg
     2461 gaagtcctgc ctcccttgct gaaggctgag agcatctatc tgaacggtct gacaccacac
     2521 tgtgctggtg aggacatagc tgtcattgag aacatgaagt gactcgagct caagcttcga
     2581 attctgcagt cgacggtacc gcgggcccgg gatccgcccc tctccctccc ccccccctaa
     2641 cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc
     2701 caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac
     2761 gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt
     2821 gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg
     2881 caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata
     2941 agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga
     3001 aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt
     3061 accccattgt atgggatctg atctggggcc tcggtacaca tgctttacat gtgtttagtc
     3121 gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac
     3181 acgatgataa tatggccaca accatggtga gcaagggcga ggagctgttc accggggtgg
     3241 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     3301 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     3361 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     3421 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     3481 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     3541 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     3601 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     3661 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     3721 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     3781 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     3841 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     3901 gcatggacga gctgtacaag taaagcggcc gcgactctag atcataatca gccataccac
     3961 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     4021 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     4081 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4141 tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa
     4201 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     4261 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     4321 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     4381 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     4441 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     4501 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     4561 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     4621 agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4681 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4741 aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     4801 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4861 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4921 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4981 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     5041 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     5101 aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg
     5161 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     5221 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     5281 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
     5341 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     5401 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     5461 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     5521 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     5581 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     5641 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg
     5701 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     5761 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     5821 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     5881 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     5941 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     6001 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     6061 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     6121 tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac
     6181 ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt
     6241 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
     6301 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
     6361 agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat
     6421 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6481 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6541 gatcaaagga tcttc
//
