LOCUS       sequence-215121-        5748 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 5748)
  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 5748)
  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: CaM12(D20A/D56A)/pIRES2-eGFP
FEATURES             Location/Qualifiers
     source          1..5748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    join(1107..1681,1682..1684,1685..1693)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1274..1291)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1501..1520
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1694..1696,1697..1699,1700..2413)
                     /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(1694..1696,1697..1699,1700..2410)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1739..1760)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2000..2019)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2347..2368
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2536..2657
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2573..2592)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2627..2646
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2664..3119)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2801..2822)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3013..3032
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3146..3250
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3252..3609
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3252..3443
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3275..3295)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3460..3595
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3644..4438
                     /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             3644..4438
                     /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(3698..3717)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4308..4327
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4626..4645)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4670..4717
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5046..5634
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5535..5554
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagat ggctgaccaa ctgacagaag agcagattgc agagttcaaa
      661 gaagccttct cattattcgc caaggatggg gacggcacca tcaccacaaa ggaacttggc
      721 accgttatga ggtcgcttgg acaaaaccca acggaagcag aattgcagga tatgatcaat
      781 gaagtcgctg ctgatggcaa tggaacgatt gactttcctg aatttcttac tatgatggct
      841 agaaaaatga aggacacaga cagcgaagag gaaatccgag aagcattccg tgtttttgac
      901 aaggatggga acggctacat cagcgctgct gaattacgtc acgtcatgac aaacctcggg
      961 gagaagttaa cagacgaaga agttgacgaa atgataaggg aagcagatat tgatggtgac
     1021 ggccaagtaa actacgaaga gtttgtacaa atgatgacag caaagtaaga attctgcagt
     1081 cgacggtacc gcgggcccgg gatccgcccc tctccctccc ccccccctaa cgttactggc
     1141 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg
     1201 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct
     1261 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca
     1321 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg
     1381 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct
     1441 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa
     1501 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt
     1561 atgggatctg atctggggcc tcggtacaca tgctttacat gtgtttagtc gaggttaaaa
     1621 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa
     1681 tatggccaca accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     1741 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     1801 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1861 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     1921 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1981 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     2041 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     2101 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     2161 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     2221 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     2281 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     2341 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     2401 gctgtacaag taaagcggcc gcgactctag atcataatca gccataccac atttgtagag
     2461 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     2521 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     2581 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     2641 ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt
     2701 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     2761 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     2821 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     2881 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     2941 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     3001 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     3061 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     3121 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3181 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3241 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     3301 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3361 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3421 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3481 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3541 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3601 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     3661 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     3721 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     3781 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     3841 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     3901 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     3961 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     4021 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     4081 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     4141 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     4201 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     4261 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     4321 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     4381 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     4441 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     4501 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4561 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     4621 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     4681 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     4741 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     4801 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     4861 ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa
     4921 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     4981 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5041 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5101 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5161 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     5221 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5281 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5341 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5401 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5461 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5521 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5581 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5641 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     5701 cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcat
//
