LOCUS       sequence-260088-        6946 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector for NaVSp1 bacterial sodium channel 
            residues 122-258. It expresses HIStag-MBP-3C-channel protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6946)
  AUTHORS   Arrigoni C, Rohaim A, Shaya D, Findeisen F, Stein RA, Nurva SR, 
            Mishra S, Mchaourab HS, Minor DL Jr
  TITLE     Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated 
            Channel Activation.
  JOURNAL   Cell. 2016 Feb 25;164(5):922-36. doi: 10.1016/j.cell.2016.02.001.
  PUBMED    26919429
REFERENCE   2  (bases 1 to 6946)
  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.cell.2016.02"; journalName: "Cell"; date: "2016-02-25- 
            25"; volume: "164"; issue: "5"; pages: "922-36"
COMMENT     Sequence Label: HM3C NaVSp1_p pET24b
FEATURES             Location/Qualifiers
     source          1..6946
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(203..220)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(650..673)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(740..1840)
                     /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     complement(743..766)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(1850..1867)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1887..1909
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1924..1948
                     /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)."
     primer_bind     complement(1948..1967)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1949..1967)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2051..2070)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2276..2353
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2354..3436
                     /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     complement(2373..2392)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4245..4436
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4430..4452)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4538..4677
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4593..4612
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4692..4709)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4863..5451)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4943..4962)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5562..5581)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5573..6388
                     /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             5573..6388
                     /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     complement(5646..5665)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6480..6935)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6617..6638)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6829..6848
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagttaacag catggaccac
      181 agcagtcgac cgagcggccg ccgctaccac ggggaaccaa cccttattac tttttggttt
      241 caccaagcgc attcagccgc tggtcgatct gttccaggcg ggccagaacc tcgtcccgat
      301 aggcatcggt ccgctcgcca tcctcggcgt gatgagcgtc ctgcatcgag ttcacgatca
      361 gaccgaccag caggttaacc accgcaaatg tggtgatcat gatgaagggg acaaagaaca
      421 gccaggcata gggatagacc tgcatcaccg ggcgaacgat gcccatcgac cagctttcca
      481 gcgtcatgat ctggaacagc gtataggccg acagacccag atcgccgaac cagtcgggaa
      541 aggaggcggc gaacagcttg gtcgcgatca ccgcgccgat gtagaagatg atggtcatca
      601 acagaaacac gctggccatg ccgggcagcg cagtgatgaa ggggctgctc ggtccttgaa
      661 acaaaacttc caaagatccg ccatcgatgg agaattcgtt gttgttattg ttattgttgt
      721 tgttgttcga gctcgaatta gtctgcgcgt ctttcagggc ttcatcgaca gtctgacgac
      781 cgctggcggc gttgatcacc gcagtacgca cggcatacca gaaagcggac atctgcggga
      841 tgttcggcat gatttcacct ttctgggcgt tttccatagt ggcggcaata cgtggatctt
      901 tcgccaactc ttcctcgtaa gacttcagcg ctacggcacc cagcggtttg tctttattaa
      961 ccgcttccag accttcatca gtcagcagat agttttcgag gaactctttt gccagctctt
     1021 tgttcggact ggcggcgtta atacctgcgc tcagcacgcc aacgaacggt ttggatggtt
     1081 gacccttgaa ggtcggcagt accgttacac cataattcac tttgctggtg tcgatgttgg
     1141 accatgccca cgggccgttg atggtcatcg ctgtttcgcc tttattaaag gcagcttctg
     1201 cgatggagta atcggtgtct gcattcatgt gtttgttttt aatcaggtca accaggaagg
     1261 tcagacccgc tttcgcgcca gcgttatcca cgcccacgtc tttaatgtcg tacttgccgt
     1321 tttcatactt gaacgcataa cccccgtcag cagcaatcag cggccaggtg aagtacggtt
     1381 cttgcaggtt gaacatcagc gcgctcttac ctttcgcttt cagttcttta tccagcgccg
     1441 ggatctcttc ccaggttttt ggcgggttcg gcagcagatc tttgttataa atcagcgata
     1501 acgcttcaac agcgatcggg taagcaatca gcttgccgtt gtaacgtacg gcatcccagg
     1561 taaacggata cagcttgtcc tggaacgctt tgtccggggt gatttcagcc aacaggccag
     1621 attgagcgta gccaccaaag cggtcgtgtg cccagaagat aatgtcaggg ccatcgccag
     1681 ttgccgcaac ctgtgggaat ttctcttcca gtttatccgg atgctcaacg gtgactttaa
     1741 ttccggtatc tttctcgaat ttcttaccga cttcagcgag accgttatag cctttatcgc
     1801 cgttaatcca gattaccagt ttaccttctt cgattttcat agaagaacca tggtgatggt
     1861 gatggtgaga agaacccata tgtatatctc cttcttaaag ttaaacaaaa ttatttctag
     1921 aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc
     1981 gagatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
     2041 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     2101 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     2161 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     2221 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac
     2281 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     2341 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     2401 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     2461 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     2521 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     2581 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     2641 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     2701 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     2761 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     2821 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     2881 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     2941 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     3001 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     3061 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     3121 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac
     3181 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
     3241 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     3301 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     3361 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     3421 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg
     3481 gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
     3541 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg
     3601 tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga
     3661 tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg
     3721 gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac
     3781 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta
     3841 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa
     3901 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg
     3961 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct
     4021 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga
     4081 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta
     4141 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta
     4201 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa
     4261 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac
     4321 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg
     4381 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     4441 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     4501 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     4561 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt
     4621 gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     4681 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4741 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4801 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4861 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4921 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4981 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5041 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5101 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5161 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5221 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5281 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     5341 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5401 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5461 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5521 gtcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     5581 tattcaacgg gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt
     5641 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt
     5701 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     5761 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     5821 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccctgg
     5881 gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     5941 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     6001 cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     6061 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     6121 taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     6181 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     6241 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     6301 acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt
     6361 tcatttgatg ctcgatgagt ttttctaaga attaattcat gagcggatac atatttgaat
     6421 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     6481 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     6541 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     6601 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     6661 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     6721 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     6781 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     6841 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     6901 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgcca
//
