LOCUS       sequence-215037-        7050 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7050)
  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 7050)
  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 AD (325-645, C643A, Delta 
            368-492)/I346D/pET...
FEATURES             Location/Qualifiers
     source          1..7050
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..27
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     111..130
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        111..129
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    130..154
                     /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)."
     RBS             169..191
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             210..227
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             237..1337
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1311..1334
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1392..1412
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2025..2042
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2095..2113)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2109..2156
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2193..2648
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2280..2299)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2490..2511
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2740..3555)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2740..3555)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3463..3482
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3547..3566
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3677..4265
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4166..4185
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4419..4436
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4451..4590
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4516..4535)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4676..4698
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4692..4883)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5692..6774)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6736..6755
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6775..6852)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
       61 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
      121 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
      181 taagaaggag atataccatg ggttcttctc accatcacca tcaccatggt tcttctatga
      241 aaatcgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg
      301 ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg
      361 ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct
      421 tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc
      481 cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg
      541 gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc
      601 tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag
      661 cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga
      721 ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg
      781 tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa
      841 acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg
      901 aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga
      961 attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg
     1021 tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg
     1081 aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg
     1141 ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacta
     1201 tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt
     1261 atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc
     1321 tgaaagacgc gcagactaat tcgagctcga acaacaacaa caataacaat aacaacaacc
     1381 tcgggatcga ggaaaacctg tattttcagg gccatatgaa ggtccaggag cagcaccggc
     1441 agaagcactt cgagaagcgg aggatgccgg cagccaacct cgaccaggct gcctggcgcc
     1501 tgtactccac cgatatgagc cgggcctacc tgacagccac ctggtacaag cttgcatctc
     1561 tgagactcaa accccgcacc tctgctgagg atgccccctc agaggaagta gcagaggaga
     1621 agagctacca gtgtgagctc acggtggacg acatcatgcc tgctgtgaag acagtcatcc
     1681 gctccatcag gattctcaag ttcctggtgg ccaaaaggaa attcaaggag acactgcgac
     1741 cgtacgacgt gaaggacgtc attgagcagt actcagcagg ccacctggac atgctgggcc
     1801 ggatcaagag cctgcaaact cgggtggacc aaattgtggg tcgggggccc ggggacagga
     1861 aggcccggga gaagggcgac aaggggccct ccgacgcgga ggtggtggat gaaatcagca
     1921 tgatgggacg cgtggtcaag gtggagaagc aggtgcagtc catcgagcac aagctggacc
     1981 tgctgttggg cttctattcg cgcgccctgc gctaatagct cgagcaccac caccaccacc
     2041 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     2101 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     2161 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     2221 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2281 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2341 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2401 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2461 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2521 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2581 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     2641 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2701 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     2761 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     2821 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     2881 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     2941 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     3001 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     3061 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     3121 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     3181 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     3241 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     3301 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     3361 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     3421 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     3481 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     3541 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     3601 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3661 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3721 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3781 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3841 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3901 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3961 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4021 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4081 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4141 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4201 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4261 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4321 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4381 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4441 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4501 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     4561 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     4621 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4681 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     4741 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     4801 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     4861 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     4921 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     4981 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     5041 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     5101 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     5161 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     5221 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     5281 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     5341 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     5401 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     5461 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     5521 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     5581 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     5641 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     5701 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5761 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     5821 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     5881 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     5941 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     6001 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     6061 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     6121 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     6181 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     6241 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     6301 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     6361 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     6421 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     6481 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     6541 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     6601 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     6661 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     6721 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     6781 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     6841 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     6901 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     6961 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     7021 tcatgagccc gaagtggcga gcccgatctt
//
