LOCUS       sequence-445181-        7848 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression vector for generating a tandem mTREK-1 with two
            identical monomers linked through a 20 AA flexible linker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7848)
  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 7848)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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(tandem wt-wt)
FEATURES             Location/Qualifiers
     source          1..7848
                     /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    3856..3908
                     /label=MCS
                     /note="multiple cloning site"
     misc_feature    join(3910..4484,4485..4487,4488..4496)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4077..4094)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4304..4323
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(4497..4499,4500..4502,4503..5216)
                     /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(4497..4499,4500..4502,4503..5213)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4542..4563)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4803..4822)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5150..5171
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5339..5460
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5376..5395)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5430..5449
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5467..5922)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5604..5625)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5816..5835
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5949..6053
                     /gene="bla"
                     /label=AmpR promoter
     promoter        6055..6412
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6055..6246
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6078..6098)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6263..6398
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6447..7241
                     /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             6447..7241
                     /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(6501..6520)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7111..7130
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7429..7448)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7473..7520
                     /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 ctcggaagct 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 tgcagctgtt 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 aacatgaagg cggccgctgg tagtggtgga
     2581 tcaggaggtt ccggcggctc tagtggatcg agtgggtcta tggcggcccc tgacttgctg
     2641 gatcccaagt ctgctgctca gaactccaaa ccgaggctct cattctcctc aaaacccacc
     2701 gtgcttgctt cccgggtgga gagtgactcg gccattaatg ttatgaaatg gaagacggtc
     2761 tccacgattt tcctggtggt cgtcctctac ctgatcatcg gagccacggt gttcaaggca
     2821 ttggagcagc ctcaggagat ttcccagagg accaccattg tgatccagaa gcagaccttc
     2881 atagcccagc atgcctgcgt caactccacc gagctggacg aactcatcca gcaaatagtg
     2941 gcagcaataa acgcaggaat tatcccctta ggaaacagct ccaatcaagt tagtcactgg
     3001 gacctcggaa gctctttctt ctttgctggt actgttatca caaccatagg atttggaaac
     3061 atctccccac gaactgaagg tggaaaaata ttctgcatca tctatgcctt gctgggaatt
     3121 cccctctttg gctttctact ggctggggtt ggtgatcagc taggaactat atttggaaaa
     3181 ggaattgcca aagtggaaga cacatttatt aagtggaatg ttagtcagac gaagattcgt
     3241 atcatctcca ccatcatctt catcctgttt ggctgtgtcc tctttgtggc tctccctgcg
     3301 gtcatattca agcacataga aggctggagc gccctggacg ctatctattt tgtggttatc
     3361 actctgacga ccattggatt tggagactac gtggcaggtg gatcagacat tgaatatctg
     3421 gacttctaca agcctgtggt gtggttctgg atcctcgttg ggctggccta ctttgcagct
     3481 gttctgagca tgattgggga ctggctacgg gtgatctcta agaagacgaa ggaagaggtg
     3541 ggagagttca gagcgcatgc cgctgagtgg acagccaatg tcacggccga gttcaaggaa
     3601 acgaggaggc ggctgagcgt ggagatctac gacaagttcc agcgtgccac atccgtgaag
     3661 cggaagctct ccgcagagct ggcgggcaac cacaaccagg aactgactcc gtgtcgtcgc
     3721 accctgtctg tgaaccacct gaccagcgag agggaagtcc tgcctccctt gctgaaggct
     3781 gagagcatct atctgaacgg tctgacacca cactgtgctg gtgaggacat agctgtcatt
     3841 gagaacatga agtagctcga gctcaagctt ccaattctgc agtcgacggt accgcgggcc
     3901 cgggatccgc ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata
     3961 aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt
     4021 gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct
     4081 cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc
     4141 ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga
     4201 caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc
     4261 ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt
     4321 attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg
     4381 gcctcggtac acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc
     4441 gaaccacggg gacgtggttt tcctttgaaa aacacgatga taatatggcc acaaccatgg
     4501 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     4561 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     4621 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     4681 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     4741 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     4801 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     4861 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     4921 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     4981 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     5041 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     5101 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     5161 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaagcg
     5221 gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     5281 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     5341 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     5401 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5461 aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     5521 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     5581 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     5641 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     5701 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     5761 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     5821 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     5881 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     5941 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     6001 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg
     6061 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     6121 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     6181 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     6241 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     6301 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     6361 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc
     6421 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     6481 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     6541 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     6601 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     6661 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     6721 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     6781 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     6841 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     6901 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     6961 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     7021 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     7081 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     7141 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     7201 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     7261 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     7321 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     7381 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     7441 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     7501 cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga
     7561 taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca
     7621 ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc
     7681 catagcctca ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     7741 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     7801 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
