LOCUS       sequence-215030-        6905 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 6905)
  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 6905)
  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 BD (522-645, C643A)/pET28HMT
FEATURES             Location/Qualifiers
     source          1..6905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     93..112
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        93..111
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    112..136
                     /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             151..173
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             193..210
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             220..1320
                     /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     1294..1317
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1375..1395
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1810..1842
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             1849..1866
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1919..1937)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1933..1980
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2017..2472
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2104..2123)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2314..2335
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2564..3379)
                     /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(2564..3379)
                     /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     3287..3306
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3371..3390
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3501..4089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3990..4009
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4243..4260
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4275..4414
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4340..4359)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4500..4522
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4516..4707)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5516..6598)
                     /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"
     promoter        complement(6599..6676)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
       61 tactggtttc acagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
      121 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
      181 atgggttctt ctcaccatca ccatcaccat ggttcttcta tgaaaatcga agaaggtaaa
      241 ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa
      301 ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa
      361 ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
      421 tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag
      481 gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac
      541 ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca
      601 aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg
      661 ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt
      721 tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
      781 ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca
      841 gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc
      901 aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta
      961 ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt
     1021 aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact
     1081 gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
     1141 tacgaggaag agttggcgaa agatccacgt attgccgcca ctatggaaaa cgcccagaaa
     1201 ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg
     1261 gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact
     1321 aattcgagct cgaacaacaa caacaataac aataacaaca acctcgggat cgaggaaaac
     1381 ctgtattttc agggcgccac ggtggacgac atcatgcctg ctgtgaagac agtcatccgc
     1441 tccatcagga ttctcaagtt cctggtggcc aaaaggaaat tcaaggagac actgcgaccg
     1501 tacgacgtga aggacgtcat tgagcagtac tcagcaggcc acctggacat gctgggccgg
     1561 atcaagagcc tgcaaactcg ggtggaccaa attgtgggtc gggggcccgg ggacaggaag
     1621 gcccgggaga agggcgacaa ggggccctcc gacgcggagg tggtggatga aatcagcatg
     1681 atgggacgcg tggtcaaggt ggagaagcag gtgcagtcca tcgagcacaa gctggacctg
     1741 ctgttgggct tctattcgcg cgccctgcgc taaaagcttg cggccgcact cgagatcaaa
     1801 cgggctagcc agccagaact cgccccggaa gaccccgagg atgtcgagca ccaccaccac
     1861 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1921 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1981 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     2041 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2101 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2161 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2221 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2281 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2341 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     2401 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2461 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     2521 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     2581 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     2641 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     2701 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     2761 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     2821 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     2881 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     2941 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     3001 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     3061 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     3121 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     3181 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     3241 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     3301 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     3361 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     3421 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3481 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3541 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3601 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3661 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3721 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3781 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3841 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3901 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3961 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4021 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4081 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4141 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4201 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4261 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4321 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4381 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4441 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4501 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4561 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     4621 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     4681 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     4741 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     4801 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     4861 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4921 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     4981 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5041 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5101 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5161 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     5221 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     5281 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     5341 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     5401 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     5461 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     5521 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5581 ggggagaggc ggtttgcgta ttgggcgcaa gggtggtttt tcttttcacc agtgagacgg
     5641 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     5701 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     5761 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     5821 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     5881 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     5941 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     6001 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     6061 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     6121 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     6181 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     6241 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     6301 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     6361 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     6421 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     6481 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     6541 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     6601 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     6661 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     6721 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcgca
     6781 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     6841 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     6901 tggtt
//
