LOCUS       sequence-342911-        7748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To generate Flp-In CHO-KCNQ3 stable cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7748)
  AUTHORS   Vanoye CG, Desai RR, Ji Z, Adusumilli S, Jairam N, Ghabra N, Joshi 
            N, Fitch E, Helbig KL, McKnight D, Lindy AS, Zou F, Helbig I, Cooper
            EC, George AL Jr
  TITLE     High-throughput evaluation of epilepsy-associated KCNQ2 variants 
            reveals functional and pharmacological heterogeneity.
  JOURNAL   JCI Insight. 2022 Mar 8;7(5):e156314. doi: 
            10.1172/jci.insight.156314.
  PUBMED    35104249
REFERENCE   2  (bases 1 to 7748)
  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"; journalName: "JCI 
            Insight."; date: "2022-03-8"; volume: "7(5):e156314. doi"; pages: " 
            10.1172/jci.insight.156314"
COMMENT     Sequence Label: pcDNA5FRT_KCNQ3
FEATURES             Location/Qualifiers
     source          1..7748
                     /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"
     5'UTR           896..938
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      949..958
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3700..3717)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3706..3930
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4063..4082)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4214..4261
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4269..5291
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4269..5291
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5421..5542
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5458..5477)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5512..5531
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5591..5607)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5591..5607)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5604..5626)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5615..5631
                     /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(5639..5645,5646..5663,5664..5669))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5684..5705
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5822..5839)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5993..6581)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6073..6092)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6752..7543,7544..7612))
                     /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     7375..7394
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7613..7717)
                     /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 gcttgcttgt
      901 tctttttgca gaagctcaga ataaacgctc aactttggca gatcgcgagc caccatgggg
      961 ctcaaggcgc gcagggcggc gggggcggct ggcggcggcg gcgacggggg cggcggaggc
     1021 ggcggggcgg ctaacccagc cggaggggac gcggcggcgg ccggcgacga ggagcggaaa
     1081 gtggggctgg cgcccggcga cgtggagcaa gtcaccttgg cgctcggggc cggagccgac
     1141 aaagacggga ccctgctgct ggagggcggc ggccgcgacg aggggcagcg gaggaccccg
     1201 cagggcatcg ggctcctggc caagaccccg ctgagccgcc cagtcaagag aaacaacgcc
     1261 aagtaccggc gcatccaaac tttgatctac gacgccctgg agagaccgcg gggctgggcg
     1321 ctgctttacc acgcgttggt gttcctgatt gtcctggggt gcttgattct ggctgtcctg
     1381 accacattca aggagtatga gactgtctcg ggagactggc ttctgttact ggagacattt
     1441 gctattttca tctttggagc cgagtttgct ttgaggatct gggctgctgg atgttgctgc
     1501 cgatacaaag gctggcgggg ccgactgaag tttgccagga agcccctgtg catgttggac
     1561 atctttgtgc tgattgcctc tgtgccagtg gttgctgtgg gaaaccaagg caatgttctg
     1621 gccacctccc tgcgaagcct gcgcttcctg cagatcctgc gcatgctgcg gatggaccgg
     1681 agaggtggca cctggaagct tctgggctca gccatctgtg cccacagcaa agaactcatc
     1741 acggcctggt acatcggttt cctgacactc atcctttctt catttcttgt ctacctggtt
     1801 gagaaagacg tcccagaggt ggatgcacaa ggagaggaga tgaaagagga gtttgagacc
     1861 tatgcagatg ccctgtggtg gggcctgatc acactggcca ccattggcta tggagacaag
     1921 acacccaaaa cgtgggaagg ccgtctgatt gccgccacct tttccttaat tggcgtctcc
     1981 ttttttgccc ttccagcggg catcctgggg tccgggctgg ccctcaaggt gcaggagcaa
     2041 caccgtcaga agcactttga gaaaaggagg aagccagctg ctgagctcat tcaggctgcc
     2101 tggaggtatt atgctaccaa ccccaacagg attgacctgg tggcgacatg gagattttat
     2161 gaatcagtcg tctcttttcc tttcttcagg aaagaacagc tggaggcagc atccagccaa
     2221 aagctgggtc tcttggatcg ggttcgcctt tctaatcctc gtggtagcaa tactaaagga
     2281 aagctattta cccctctgaa tgtagatgcc atagaagaaa gtccttctaa agaaccaaag
     2341 cctgttggct taaacaataa agagcgtttc cgcacggcct tccgcatgaa agcctacgct
     2401 ttctggcaga gttctgaaga tgccgggaca ggtgacccca tggcggaaga caggggctat
     2461 gggaatgact tccccatcga agacatgatc cccaccctga aggccgccat ccgagccgtc
     2521 agaattctac aattccgtct ctataaaaaa aaattcaagg agactttgag gccttacgat
     2581 gtgaaggatg tgattgagca gtattctgcc gggcatctcg acatgctttc caggataaag
     2641 taccttcaga cgagaataga tatgattttc acccctggac ctccctccac gccaaaacac
     2701 aagaagtctc agaaagggtc agcattcacc ttcccatccc agcaatctcc caggaatgaa
     2761 ccatatgtag ccagaccatc cacatcagaa atcgaagacc aaagcatgat ggggaagttt
     2821 gtaaaagttg aaagacaggt tcaggacatg gggaagaagc tggacttcct cgtggatatg
     2881 cacatgcaac acatggaacg gttgcaggtg caggtcacgg agtattaccc aaccaagggc
     2941 acctcctcgc cagctgaagc agagaagaag gaggacaaca ggtattccga tttgaaaacc
     3001 atcatctgca actattctga gacaggcccc ccggaaccac cctacagctt ccaccaggtg
     3061 accattgaca aagtcagccc ctatgggttt tttgcacatg accctgtgaa cctgccccga
     3121 gggggaccca gttctggaaa ggttcaggca actcctcctt cctcagcaac aacgtatgtg
     3181 gagaggccca cggtcctgcc tatcttgact cttctcgact cccgagtgag ctgccactcc
     3241 caggctgacc tgcagggccc ctactcggac cgaatctccc cccggcagag acgtagcatc
     3301 acgcgagaca gtgacacacc tctgtccctg atgtcggtca accacgagga gctggagagg
     3361 tctccaagtg gcttcagcat ctcccaggac agagatgatt atgtgttcgg ccccaatggg
     3421 gggtcgagct ggatgaggga gaagcggtac ctcgccgagg gtgagacgga cacagacacg
     3481 gaccccttca cgcccagcgg ctccatgcct ctgtcgtcca caggggatgg gatttctgat
     3541 tcagtatgga ccccttccaa taagcccatt taaggaattc atcgatatct cgagcatgca
     3601 tctagaggat ccactagtcc agtgtggtgg aattctgcag atatccagca cagtggcggc
     3661 cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     3721 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3781 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3841 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     3901 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     3961 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4021 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4081 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4141 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     4201 gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt
     4261 ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     4321 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
     4381 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
     4441 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
     4501 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
     4561 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
     4621 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
     4681 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
     4741 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
     4801 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
     4861 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
     4921 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
     4981 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
     5041 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
     5101 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
     5161 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
     5221 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
     5281 caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5341 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5401 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     5461 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5521 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5581 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5641 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5701 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5761 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5821 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5881 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5941 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6001 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6061 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6121 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6181 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6241 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6301 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6361 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6421 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6481 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6541 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6601 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6661 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6721 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6781 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6841 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6901 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6961 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7021 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7081 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7141 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7201 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7261 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7321 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7381 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7441 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7501 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7561 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7621 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7681 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7741 ctgacgtc
//
