LOCUS       sequence-215075-        6669 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 6669)
  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 6669)
  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: Kv7.5 AB (361-545, Delta-395-511)/pET28HMT
FEATURES             Location/Qualifiers
     source          1..6669
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     91..110
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        91..109
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    110..134
                     /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             149..171
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             190..207
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             217..1317
                     /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     1291..1314
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1372..1392
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1624..1641
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1694..1712)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1708..1755
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1792..2247
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1879..1898)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2089..2110
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2339..3154)
                     /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(2339..3154)
                     /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     3062..3081
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3146..3165
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3276..3864
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3765..3784
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4018..4035
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4050..4189
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4115..4134)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4275..4297
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4291..4482)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5291..6373)
                     /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     6335..6354
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6374..6451)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6657..6669,1..7)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
       61 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
      121 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg
      181 ggttcttctc accatcacca tcaccatggt tcttctatga aaatcgaaga aggtaaactg
      241 gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc
      301 gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc
      361 ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt
      421 ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac
      481 aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg
      541 atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa
      601 acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg
      661 atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat
      721 gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc
      781 gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac
      841 accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac
      901 ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg
      961 ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac
     1021 gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct gctgactgat
     1081 gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac
     1141 gaggaagagt tggcgaaaga tccacgtatt gccgccacta tggaaaacgc ccagaaaggt
     1201 gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg
     1261 atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagactaat
     1321 tcgagctcga acaacaacaa caataacaat aacaacaacc tcgggatcga ggaaaacctg
     1381 tattttcagg gccatatggc tagcaaacac tttgagaaaa gaaggaaccc agctgccaac
     1441 ctcattcagt gtgtttggcg tagttacgca gctgatgaga aatctgtttc cattgcaacc
     1501 tggaagaagc ttgaagacct caccccacca cttaaaactg tcattcgagc tatcagaatt
     1561 atgaaatttc atgttgcaaa acggaagttt aaggaaacat tacgtccata tgattgactc
     1621 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     1681 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1741 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     1801 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1861 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1921 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1981 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     2041 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     2101 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     2161 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2221 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     2281 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     2341 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     2401 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     2461 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     2521 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     2581 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     2641 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     2701 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     2761 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     2821 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     2881 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     2941 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     3001 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     3061 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     3121 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     3181 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     3241 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3301 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3361 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3421 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3481 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3541 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3601 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3661 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3721 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3781 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3841 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3901 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3961 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     4021 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     4081 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4141 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     4201 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4261 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4321 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     4381 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     4441 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     4501 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     4561 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     4621 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     4681 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     4741 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     4801 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     4861 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     4921 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     4981 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     5041 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     5101 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     5161 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     5221 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     5281 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5341 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     5401 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     5461 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     5521 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     5581 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     5641 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     5701 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     5761 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     5821 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     5881 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     5941 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     6001 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     6061 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     6121 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     6181 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     6241 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     6301 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     6361 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     6421 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     6481 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     6541 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     6601 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     6661 atgtcggcg
//
