LOCUS       sequence-215073-        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)/I346A/pET...
FEATURES             Location/Qualifiers
     source          1..7050
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     62..81
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        62..80
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    81..105
                     /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             120..142
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             161..178
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             188..1288
                     /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     1262..1285
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1343..1363
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1976..1993
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2046..2064)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2060..2107
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2144..2599
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2231..2250)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2441..2462
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2691..3506)
                     /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(2691..3506)
                     /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     3414..3433
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3498..3517
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3628..4216
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4117..4136
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4370..4387
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4402..4541
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4467..4486)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4627..4649
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4643..4834)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5643..6725)
                     /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     6687..6706
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6726..6803)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7009..7028
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
       61 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
      121 ttgtttaact ttaagaagga gatataccat gggttcttct caccatcacc atcaccatgg
      181 ttcttctatg aaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta
      241 taacggtctc gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt
      301 tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc
      361 tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc
      421 tgaaatcacc ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt
      481 acgttacaac ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta
      541 taacaaagat ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa
      601 agaactgaaa gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac
      661 ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga
      721 cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga
      781 cctgattaaa aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt
      841 taataaaggc gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac
      901 cagcaaagtg aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc
      961 gttcgttggc gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcaaa
     1021 agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa
     1081 accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat
     1141 tgccgccact atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc
     1201 cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt
     1261 cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg aacaacaaca acaataacaa
     1321 taacaacaac ctcgggatcg aggaaaacct gtattttcag ggccatatga aggtccagga
     1381 gcagcaccgg cagaagcact tcgagaagcg gaggatgccg gcagccaacc tcgcccaggc
     1441 tgcctggcgc ctgtactcca ccgatatgag ccgggcctac ctgacagcca cctggtacaa
     1501 gcttgcatct ctgagactca aaccccgcac ctctgctgag gatgccccct cagaggaagt
     1561 agcagaggag aagagctacc agtgtgagct cacggtggac gacatcatgc ctgctgtgaa
     1621 gacagtcatc cgctccatca ggattctcaa gttcctggtg gccaaaagga aattcaagga
     1681 gacactgcga ccgtacgacg tgaaggacgt cattgagcag tactcagcag gccacctgga
     1741 catgctgggc cggatcaaga gcctgcaaac tcgggtggac caaattgtgg gtcgggggcc
     1801 cggggacagg aaggcccggg agaagggcga caaggggccc tccgacgcgg aggtggtgga
     1861 tgaaatcagc atgatgggac gcgtggtcaa ggtggagaag caggtgcagt ccatcgagca
     1921 caagctggac ctgctgttgg gcttctattc gcgcgccctg cgctaatagc tcgagcacca
     1981 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     2041 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     2101 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     2161 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2221 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2281 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2341 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2401 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2461 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2521 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     2581 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2641 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     2701 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     2761 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     2821 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     2881 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     2941 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     3001 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     3061 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     3121 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     3181 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     3241 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     3301 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     3361 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     3421 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     3481 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     3541 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     3601 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     3661 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3721 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     3781 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3841 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3901 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3961 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4021 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4081 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4141 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4201 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4261 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4321 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     4381 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4441 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4501 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     4561 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4621 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     4681 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     4741 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     4801 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     4861 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     4921 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4981 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     5041 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     5101 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     5161 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     5221 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     5281 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     5341 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     5401 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     5461 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     5521 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     5581 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     5641 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5701 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     5761 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     5821 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     5881 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     5941 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     6001 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     6061 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     6121 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     6181 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     6241 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     6301 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     6361 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     6421 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     6481 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     6541 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     6601 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     6661 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     6721 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     6781 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     6841 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     6901 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     6961 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     7021 gatataggcg ccagcaaccg cacctgtggc
//
