LOCUS       sequence-9495-ex        6996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6996)
  AUTHORS   Suh BC, Inoue T, Meyer T, Hille B
  TITLE     Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion 
            channels.
  JOURNAL   Science. 2006 Dec 1. 314(5804):1454-7.
  PUBMED    16990515
REFERENCE   2  (bases 1 to 6996)
  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: "Science. 
            2006 Dec 1. 314(5804):1454-7."
COMMENT     Sequence Label: CF-PIPKi
FEATURES             Location/Qualifiers
     source          1..6996
                     /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"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1342..1662
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     polyA_signal    3784..3905
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3821..3840)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3875..3894
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3912..4367)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4049..4070)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4261..4280
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4394..4498
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4500..4857
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4500..4691
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4523..4543)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4708..4843
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4892..5686
                     /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             4892..5686
                     /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(4946..4965)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5556..5575
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5874..5893)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5918..5965
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6294..6882
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6783..6802
                     /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 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tggggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac atcagccaca acgtctatat caccgccgac
     1081 aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaaga gtgctggtgg tggagtgcag gtggaaacca tctccccagg agacgggcgc
     1381 accttcccca agcgcggcca gacctgcgtg gtgcactaca ccgggatgct tgaagatgga
     1441 aagaaatttg attcctcccg ggacagaaac aagcccttta agtttatgct aggcaagcag
     1501 gaggtgatcc gaggctggga agaaggggtt gcccagatga gtgtgggtca gagagccaaa
     1561 ctgactatat ctccagatta tgcctatggt gccactgggc acccaggcat catcccacca
     1621 catgccactc tcgtcttcga tgtggagctt ctaaaactgg aaagtgctgg tggtagtgct
     1681 ggtggtagtg ctggtggtag tgctggtggt agtgctggtg gtcctcgagc tcaagcttcg
     1741 aattcgatgg agctagaggt gccggacgag gcggagagcg ccgaagcggg ggccgtgacg
     1801 gcggaagcgg cctggtctgc ggagagtggg gcggcggcag gtatgaccca gaagaaggct
     1861 ggcctcgcag aggccccctt ggtgacaggg cagccaggcc ctggccatgg gaagaagctg
     1921 ggccaccgag gcgtggatgc gtcgggagag actacgtata agaagaccac ctcgtccacc
     1981 ctgaagggcg ccatccagct ggggatcggg tacacggtgg gcaacctgag ctctaagccg
     2041 gagcgggacg tgctcatgca ggacttctac gtgatggaga gcatcttctt ccccagtgaa
     2101 gggagcaact tcacccctgc ccaccacttc caggatttcc gcttcaagac ctatgcacct
     2161 gttgccttcc gctacttccg ggagctcttt ggcatccgtc cagatgacta tttgtactcc
     2221 ctgtgcaatg agccactcat cgagctctcc aaccccgggg ccagcggctc tgtcttctac
     2281 gtcaccagcg acgacgagtt catcatcaag actgtcatgc acaaggaggc agagttcctg
     2341 cagaagctgc tgcctggcta ctacatgaat ctcaaccaga acccacggac gctgctgccc
     2401 aagttctatg ggctgtactg cgtgcagtct ggtggcaaga acatccgcgt ggtggtcatg
     2461 aacaatgtgc tgccccgtgt cgtcaaaatg caccttaagt tcgccctcaa gggctccacg
     2521 tacaagcgca gggccagcaa gaaggagaag gagaagagcc tgcccaccta caaggacctg
     2581 gacttcatgc aggacatgcc cgaggggctg ctgctggact ccgacacctt tggcgccctg
     2641 gtcaagacgc tgcagcgaga ctgcctggtg ctggagagct tcaagataat ggactacagc
     2701 ctgctgctgg gcgtgcacaa catcgatcag caggagcgag agcgccaggc cgagggcgcc
     2761 cagagcaagg cggatgagaa gcggcccgtg gcccagaagg ctctgtattc cacggccatg
     2821 gagtctatcc agggcggagc tgcccgtggg gaggccattg agacagatga cacgatgggt
     2881 gggattccag cagtgaatgg gcgcggggag cgactgctcc tccacatcgg gatcattgat
     2941 attctgcagt cctacaggtt catcaagaag ttagaacaca cctggaaggc cctcgtccat
     3001 gatggggaca ctgtctcagt ccaccggccc agcttctatg cagagcgctt cttcaagttc
     3061 atgagcagca cggtgttcga ggagagttcc tccctgaagt cctctccatc caagaaagga
     3121 cgtggtgccc tgctggcggt caaacccctg gggcccactg ctgccttctc agccagccag
     3181 atccccagcg agagagagga tgtgcagtat gacctgcggg gggcccgcag ctaccccacg
     3241 cttgaggatg aaggccggcc tgacctcctg ccctgcaccc caccgtcctt tgaggaagcc
     3301 accacagcct ccatcgccac caccctgtcg tccacctccc tctccatccc agagcggtcc
     3361 ccttcagata catcggagca gccccggtac aggcggcgca cgcagtcttc aggccaggat
     3421 ggccggcccc aggaggagcc ccatgcggaa gacctgcaga agataactgt gcaggtggag
     3481 ccagtgtgcg gtgtgggggt tgtccccaag gaggagggtg caggagtgga ggtcccccca
     3541 tgtggggcat cggctgcagc ctctgtggaa atagacgctg ccagccaggc ctcagagcct
     3601 gccagccagg cctcagatga ggaggatgca ccctctacag acatctattt ttaaggatcc
     3661 accggatcta gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct
     3721 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     3781 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3841 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     3901 tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     3961 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     4021 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     4081 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     4141 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     4201 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     4261 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     4321 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     4381 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4441 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc
     4501 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     4561 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     4621 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4681 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4741 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     4801 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat
     4861 cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     4921 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     4981 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     5041 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     5101 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     5161 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     5221 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     5281 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     5341 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     5401 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     5461 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     5521 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     5581 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     5641 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     5701 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     5761 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     5821 cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca
     5881 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     5941 acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     6001 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     6061 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     6121 cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa
     6181 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6241 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6301 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6361 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6421 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     6481 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6541 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6601 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6661 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6721 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6781 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6841 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6901 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     6961 cctgattctg tggataaccg tattaccgcc atgcat
//
