LOCUS       sequence-215153-        7731 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Electrophysiology in HEK cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7731)
  AUTHORS   Chang A, Abderemane-Ali F, Hura GL, Rossen ND, Gate RE, Minor DL Jr.
  TITLE     A Calmodulin C-Lobe Ca(2+)-Dependent Switch Governs Kv7 Channel 
            Function.
  JOURNAL   Neuron. 2018 Feb 21;97(4):836-852.e6. doi: 
            10.1016/j.neuron.2018.01.035. Epub 2018 Feb 8.
  PUBMED    29429937
REFERENCE   2  (bases 1 to 7731)
  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.neuron.2018.01.035"; journalName: "Neuron"; date: 
            "2018-02-21- 21"; volume: "97"; issue: "4"; pages: "836-852.e6"
COMMENT     Sequence Label: Kv7.4 (1-645, Delta-368-523)/S536D/L540D/HA 
            tag/pcD...
FEATURES             Location/Qualifiers
     source          1..7731
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             938..964
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2462..2482
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(2483..2485,2486..3196)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2483..2485,2486..3196)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2525..2546)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2786..2805)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3133..3154
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3197..3220
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     promoter        complement(3284..3302)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3285..3302)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3322..3339)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3328..3552
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3598..4026
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3685..3704)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3895..3916
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4035..4055)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4040..4369
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4220..4355
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4436..5230
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4490..4509)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5100..5119
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5404..5525
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5441..5460)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5495..5514
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5574..5590)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5574..5590)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5587..5609)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5598..5614
                     /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(5622..5628,5629..5646,5647..5652))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5667..5688
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5805..5822)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5976..6564)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6056..6075)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6735..7526,7527..7595))
                     /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     7358..7377
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7596..7700)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccttgggtac ttccaatcca tttgatgtat ccatatgatg ttccagatta
      961 tgctgccgag gcccccccgc gccgcctcgg cctgggtccc ccgcccgggg acgccccccg
     1021 cgcggagcta gtggcgctca cggccgtgca gagcgaacag ggcgaggcgg gcgggggcgg
     1081 ctccccgcgc cgcctcggcc tcctgggcag ccccctgccg ccgggcgcgc ccctccctgg
     1141 gccgggctcc ggctcgggct ccgcctgcgg ccagcgctcc tcggccgcgc acaagcgcta
     1201 ccgccgcctg cagaactggg tctacaacgt gctggagcgg ccccgcggct gggccttcgt
     1261 ctaccacgtc ttcatatttt tgctggtctt cagctgtctg gtgctgtctg tgctgtccac
     1321 tatccaggag caccaggaac ttgccaacga gtgtctcctc atcttggaat tcgtgatgat
     1381 cgtggttttc ggcttggagt acatcgtccg ggtctggtcc gccggatgct gctgccgcta
     1441 ccgaggatgg cagggtcgct tccgctttgc cagaaagccc ttctgtgtca tcgacttcat
     1501 cgtgttcgtg gcctcggtgg ccgtcatcgc cgcgggtacc cagggcaaca tcttcgccac
     1561 gtccgcgctg cgcagcatgc gcttcctgca gatcctgcgc atggtgcgca tggaccgccg
     1621 cggcggcacc tggaagctgc tgggctcagt ggtctacgcg catagcaagg agctgatcac
     1681 cgcctggtac atcgggttcc tggtgctcat cttcgcctcc ttcctggtct acctggccga
     1741 gaaggacgcc aactccgact tctcctccta cgccgactcg ctctggtggg ggacgattac
     1801 attgacaacc atcggctatg gtgacaagac accgcacaca tggctgggca gggtcctggc
     1861 tgctggcttc gccttactgg gcatctcttt ctttgccctg cctgccggca tcctaggctc
     1921 cggctttgcc ctgaaggtcc aggagcagca ccggcagaag cacttcgaga agcggaggat
     1981 gccggcagcc aacctcatcc aggctgcctg gcgcctgtac tccaccgata tgagccgggc
     2041 ctacctgaca gccacctggt acaagcttga cgacatcatg cctgctgtga agacagtcat
     2101 ccgcgacatc aggattgaca agttcctggt ggccaaaagg aaattcaagg agacactgcg
     2161 accgtacgac gtgaaggacg tcattgagca gtactcagca ggccacctgg acatgctggg
     2221 ccggatcaag agcctgcaaa ctcgggtgga ccaaattgtg ggtcgggggc ccggggacag
     2281 gaaggcccgg gagaagggcg acaaggggcc ctccgacgcg gaggtggtgg atgaaatcag
     2341 catgatggga cgcgtggtca aggtggagaa gcaggtgcag tccatcgagc acaagctgga
     2401 cctgctgttg ggcttctatt cgcgcgccct gcgctaaggc aaattgggat taattaatct
     2461 cgaaaacctg tacttccaat ccgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     2521 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     2581 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2641 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     2701 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2761 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2821 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2881 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2941 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     3001 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     3061 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     3121 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     3181 ggacgagctg tacaagcatc accatcacca tcaccatcac taaggatcct gcagatatcc
     3241 atcacactgg cggccgctcg agcatgcatc tagagggccc tattctatag tgtcacctaa
     3301 atgctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     3361 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     3421 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     3481 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     3541 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     3601 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3661 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3721 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3781 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     3841 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3901 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3961 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4021 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     4081 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     4141 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     4201 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     4261 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     4321 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     4381 agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat
     4441 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4501 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4561 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     4621 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4681 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4741 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4801 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4861 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4921 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     4981 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     5041 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     5101 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     5161 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     5221 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     5281 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     5341 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     5401 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     5461 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     5521 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     5581 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5641 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5701 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5761 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5821 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5881 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5941 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     6001 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     6061 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     6121 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     6181 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6241 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6301 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6361 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6421 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6481 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6541 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6601 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6661 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6721 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6781 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6841 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6901 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6961 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     7021 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     7081 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     7141 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     7201 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7261 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7321 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     7381 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7441 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7501 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7561 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7621 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7681 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
