LOCUS       sequence-215071-        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)/S536A/L53...
FEATURES             Location/Qualifiers
     source          1..7050
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     90..109
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        90..108
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    109..133
                     /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             148..170
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             189..206
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             216..1316
                     /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     1290..1313
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1371..1391
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2004..2021
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2074..2092)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2088..2135
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2172..2627
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2259..2278)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2469..2490
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2719..3534)
                     /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(2719..3534)
                     /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     3442..3461
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3526..3545
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3656..4244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4145..4164
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4398..4415
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4430..4569
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4495..4514)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4655..4677
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4671..4862)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5671..6753)
                     /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     6715..6734
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6754..6831)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(7037..7050,1..6)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
       61 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
      121 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg
      181 gttcttctca ccatcaccat caccatggtt cttctatgaa aatcgaagaa ggtaaactgg
      241 taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt aagaaattcg
      301 agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa gagaaattcc
      361 cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac gaccgctttg
      421 gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg ttccaggaca
      481 agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt gcttacccga
      541 tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac ccgccaaaaa
      601 cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag agcgcgctga
      661 tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac gggggttatg
      721 cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat aacgctggcg
      781 cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg aatgcagaca
      841 ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg accatcaacg
      901 gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta acggtactgc
      961 cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg
     1021 ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg ctgactgatg
     1081 aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg
     1141 aggaagagtt ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc cagaaaggtg
     1201 aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga
     1261 tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagactaatt
     1321 cgagctcgaa caacaacaac aataacaata acaacaacct cgggatcgag gaaaacctgt
     1381 attttcaggg ccatatgaag gtccaggagc agcaccggca gaagcacttc gagaagcgga
     1441 ggatgccggc agccaacctc atccaggctg cctggcgcct gtactccacc gatatgagcc
     1501 gggcctacct gacagccacc tggtacaagc ttgcatctct gagactcaaa ccccgcacct
     1561 ctgctgagga tgccccctca gaggaagtag cagaggagaa gagctaccag tgtgagctca
     1621 cggtggacga catcatgcct gctgtgaaga cagtcatccg cgccatcagg attgccaagt
     1681 tcctggtggc caaaaggaaa ttcaaggaga cactgcgacc gtacgacgtg aaggacgtca
     1741 ttgagcagta ctcagcaggc cacctggaca tgctgggccg gatcaagagc ctgcaaactc
     1801 gggtggacca aattgtgggt cgggggcccg gggacaggaa ggcccgggag aagggcgaca
     1861 aggggccctc cgacgcggag gtggtggatg aaatcagcat gatgggacgc gtggtcaagg
     1921 tggagaagca ggtgcagtcc atcgagcaca agctggacct gctgttgggc ttctattcgc
     1981 gcgccctgcg ctaatagctc gagcaccacc accaccacca ctgagatccg gctgctaaca
     2041 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     2101 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     2161 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2221 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2281 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2341 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2401 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     2461 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2521 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2581 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     2641 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2701 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     2761 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     2821 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     2881 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     2941 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     3001 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     3061 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     3121 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     3181 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     3241 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     3301 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     3361 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     3421 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     3481 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     3541 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     3601 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3661 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3721 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3781 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3841 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3901 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3961 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4021 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4081 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4141 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4201 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4261 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4321 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4381 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     4441 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     4501 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     4561 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4621 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4681 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     4741 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     4801 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     4861 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     4921 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     4981 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     5041 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     5101 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     5161 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     5221 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     5281 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     5341 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     5401 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     5461 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     5521 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     5581 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     5641 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5701 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5761 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     5821 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     5881 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     5941 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     6001 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     6061 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     6121 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     6181 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     6241 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     6301 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     6361 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     6421 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     6481 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     6541 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     6601 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     6661 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     6721 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     6781 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     6841 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     6901 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     6961 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     7021 cccgatcttc cccatcggtg atgtcggcga
//
