LOCUS       sequence-215005-        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)/pET28HMT
FEATURES             Location/Qualifiers
     source          1..7050
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     45..64
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        45..63
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    64..88
                     /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             103..125
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             144..161
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             171..1271
                     /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     1245..1268
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1326..1346
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1959..1976
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2029..2047)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2043..2090
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2127..2582
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2214..2233)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2424..2445
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2674..3489)
                     /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(2674..3489)
                     /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     3397..3416
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3481..3500
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3611..4199
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4100..4119
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4353..4370
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4385..4524
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4450..4469)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4610..4632
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4626..4817)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5626..6708)
                     /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     6670..6689
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6709..6786)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6992..7011
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
       61 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
      121 ggagatatac catgggttct tctcaccatc accatcacca tggttcttct atgaaaatcg
      181 aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt ctcgctgaag
      241 tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat ccggataaac
      301 tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt atcttctggg
      361 cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc accccggaca
      421 aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac aacggcaagc
      481 tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa gatctgctgc
      541 cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg aaagcgaaag
      601 gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg ctgattgctg
      661 ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa gacgtgggcg
      721 tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt aaaaacaaac
      781 acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa ggcgaaacag
      841 cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa gtgaattatg
      901 gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga
      961 gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc ctcgaaaact
     1021 atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag
     1081 cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc actatggaaa
     1141 acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc tggtatgccg
     1201 tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag
     1261 acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac aacctcggga
     1321 tcgaggaaaa cctgtatttt cagggccata tgaaggtcca ggagcagcac cggcagaagc
     1381 acttcgagaa gcggaggatg ccggcagcca acctcatcca ggctgcctgg cgcctgtact
     1441 ccaccgatat gagccgggcc tacctgacag ccacctggta caagcttgca tctctgagac
     1501 tcaaaccccg cacctctgct gaggatgccc cctcagagga agtagcagag gagaagagct
     1561 accagtgtga gctcacggtg gacgacatca tgcctgctgt gaagacagtc atccgctcca
     1621 tcaggattct caagttcctg gtggccaaaa ggaaattcaa ggagacactg cgaccgtacg
     1681 acgtgaagga cgtcattgag cagtactcag caggccacct ggacatgctg ggccggatca
     1741 agagcctgca aactcgggtg gaccaaattg tgggtcgggg gcccggggac aggaaggccc
     1801 gggagaaggg cgacaagggg ccctccgacg cggaggtggt ggatgaaatc agcatgatgg
     1861 gacgcgtggt caaggtggag aagcaggtgc agtccatcga gcacaagctg gacctgctgt
     1921 tgggcttcta ttcgcgcgcc ctgcgctaat agctcgagca ccaccaccac caccactgag
     1981 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     2041 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     2101 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     2161 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2221 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2281 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2341 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2401 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2461 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2521 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     2581 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2641 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
     2701 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     2761 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     2821 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     2881 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     2941 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     3001 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     3061 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     3121 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     3181 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     3241 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     3301 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     3361 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     3421 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     3481 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     3541 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3601 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3661 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3721 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     3781 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3841 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3901 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3961 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4021 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4081 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4141 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4201 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4261 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4321 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4381 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     4441 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     4501 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     4561 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4621 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     4681 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     4741 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     4801 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     4861 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     4921 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     4981 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     5041 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     5101 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     5161 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     5221 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     5281 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     5341 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     5401 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     5461 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     5521 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     5581 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     5641 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5701 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     5761 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     5821 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     5881 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     5941 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     6001 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     6061 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     6121 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     6181 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     6241 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     6301 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     6361 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     6421 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     6481 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     6541 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     6601 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     6661 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     6721 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     6781 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     6841 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     6901 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     6961 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     7021 ccgcacctgt ggcgccggtg atgccggcca
//
