LOCUS       sequence-448327-        7124 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Neuronal expression IRES-containing bicistronic vector for 
            generating mTREK-1 (S131C/A286F) + EGFP under the hSyn promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7124)
  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 7124)
  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: pCDNA3.1(+)-hSyn-HA-mTREK-1(S131C/A286F)-IRES-GFP
FEATURES             Location/Qualifiers
     source          1..7124
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(182..973,974..1042))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     805..824
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1043..1147)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1174..1629
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1261..1280)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1471..1492
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1755..1777
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1769..1786
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1770..1786
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1994..2441
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             2465..2491
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    3725..3777
                     /label=MCS
                     /note="multiple cloning site"
     misc_feature    join(3779..4353,4354..4356,4357..4365)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3946..3963)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4173..4192
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(4366..4368,4369..4371,4372..5085)
                     /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(4366..4368,4369..4371,4372..5082)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4411..4432)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4672..4691)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5019..5040
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5094..5716
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5293..5312)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(5740..5760)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5745..6074
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5925..6060
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6112..6131)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6113..6131)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6145..6161)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6145..6161)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6158..6180)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6169..6185
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6193..6199,6200..6217,6218..6223))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6238..6259
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6376..6393)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6547..7124,1..11))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6627..6646)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
       61 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
      121 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
      181 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
      241 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
      301 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
      361 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
      421 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
      481 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
      541 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
      601 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
      661 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
      721 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
      781 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
      841 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
      901 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
      961 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     1021 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     1081 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     1141 ttccgcgcac atttccccga aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg
     1201 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1261 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1321 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1381 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1441 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1501 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1561 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1621 ttacaatttc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     1681 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     1741 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgcca agctgatcta
     1801 tacattgaat caatattggc aattagccat attagtcatt ggttatatag cataaatcaa
     1861 tattggctat tggccattgc atacgttgta tctatatcat aatatgtaca tttatattgg
     1921 ctcatgtcca atatgaccgc catgttgaca ttgattattg actagactcg agctgcagag
     1981 ggccctgcgt atgagtgcaa gtgggtttta ggaccaggat gaggcggggt gggggtgcct
     2041 acctgacgac cgaccccgac ccactggaca agcacccaac ccccattccc caaattgcgc
     2101 atcccctatc agagaggggg aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag
     2161 cttcagcacc gcggacagtg ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca
     2221 gcactgaagg cgcgctgacg tcactcgccg gtcccccgca aactcccctt cccggccacc
     2281 ttggtcgcgt ccgcgccgcc gccggcccag ccggaccgca ccacgcgagg cgcgagatag
     2341 gggggcacgg gcgcgaccat ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg
     2401 cggtgggcag cggaggagtc gtgtcgtgcc tgagagcgca gtcgaattca gatctggtac
     2461 catgtaccca tacgacgtgc cagactacgc tgcggcccct gacttgctgg atcccaagtc
     2521 tgctgctcag aactccaaac cgaggctctc attctcctca aaacccaccg tgcttgcttc
     2581 ccgggtggag agtgactcgg ccattaatgt tatgaaatgg aagacggtct ccacgatttt
     2641 cctggtggtc gtcctctacc tgatcatcgg agccacggtg ttcaaggcat tggagcagcc
     2701 tcaggagatt tcccagagga ccaccattgt gatccagaag cagaccttca tagcccagca
     2761 tgcctgcgtc aactccaccg agctggacga actcatccag caaatagtgg cagcaataaa
     2821 cgcaggaatt atccccttag gaaacagctc caatcaagtt agtcactggg acctcggatg
     2881 ctctttcttc tttgctggta ctgttatcac aaccatagga tttggaaaca tctccccacg
     2941 aactgaaggt ggaaaaatat tctgcatcat ctatgccttg ctgggaattc ccctctttgg
     3001 ctttctactg gctggggttg gtgatcagct aggaactata tttggaaaag gaattgccaa
     3061 agtggaagac acatttatta agtggaatgt tagtcagacg aagattcgta tcatctccac
     3121 catcatcttc atcctgtttg gctgtgtcct ctttgtggct ctccctgcgg tcatattcaa
     3181 gcacatagaa ggctggagcg ccctggacgc tatctatttt gtggttatca ctctgacgac
     3241 cattggattt ggagactacg tggcaggtgg atcagacatt gaatatctgg acttctacaa
     3301 gcctgtggtg tggttctgga tcctcgttgg gctggcctac tttttcgctg ttctgagcat
     3361 gattggggac tggctacggg tgatctctaa gaagacgaag gaagaggtgg gagagttcag
     3421 agcgcatgcc gctgagtgga cagccaatgt cacggccgag ttcaaggaaa cgaggaggcg
     3481 gctgagcgtg gagatctacg acaagttcca gcgtgccaca tccgtgaagc ggaagctctc
     3541 cgcagagctg gcgggcaacc acaaccagga actgactccg tgtcgtcgca ccctgtctgt
     3601 gaaccacctg accagcgaga gggaagtcct gcctcccttg ctgaaggctg agagcatcta
     3661 tctgaacggt ctgacaccac actgtgctgg tgaggacata gctgtcattg agaacatgaa
     3721 gtgactcgag ctcaagcttc gaattctgca gtcgacggta ccgcgggccc gggatccgcc
     3781 cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg
     3841 cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga
     3901 aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa
     3961 tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa
     4021 caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct
     4081 gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg
     4141 ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg
     4201 ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtaca
     4261 catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg
     4321 acgtggtttt cctttgaaaa acacgatgat aatatggcca caaccatggt gagcaagggc
     4381 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     4441 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     4501 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     4561 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     4621 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     4681 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     4741 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     4801 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     4861 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     4921 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     4981 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     5041 accgccgccg ggatcactct cggcatggac gagctgtaca agtaaaggat cccgggtggc
     5101 atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca
     5161 ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata
     5221 atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta
     5281 gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg
     5341 caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag ttgttgggat
     5401 tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca
     5461 ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc
     5521 ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgattttaa
     5581 aataactata ccagcaggag gacgtccaga cacagcatag gctacctggc catgcccaac
     5641 cggtgggaca tttgagttgc ttgcttggca ctgtcctctc atgcgttggg tccactcagt
     5701 agatgcctgt tgaattgggt acgcggccag cttggctgtg gaatgtgtgt cagttagggt
     5761 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     5821 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     5881 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     5941 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     6001 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     6061 taggcttttg caaaaagctc ctcgaggaac tgaaaaacca gaaagttaat tccctatagt
     6121 gagtcgtatt aaattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct
     6181 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     6241 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6301 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6361 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     6421 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     6481 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6541 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6601 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6661 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6721 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6781 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6841 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6901 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6961 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     7021 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     7081 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaa
//
