LOCUS Exported 7101 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Electrophysiology in mammalian cells: Mammalian expression of voltage gated sodium channel and GFP. ACCESSION . VERSION . KEYWORDS His-NavMs SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7101) AUTHORS Sula A, Booker J, Ng LC, Naylor CE, DeCaen PG, Wallace BA TITLE The complete structure of an activated open sodium channel. JOURNAL Nat Commun. 2017 Feb 16;8:14205. doi: 10.1038/ncomms14205. PUBMED 28205548 REFERENCE 2 (bases 1 to 7101) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7101 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 5..384 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 385..588 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 539..559 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" promoter 633..651 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 729..738 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 747..764 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 774..791 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" primer_bind complement(1670..1687) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1676..1787 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1955..3133 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 2186..3124 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 3081..3101 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" promoter 3150..3168 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 3257..3304 /label=EM7 promoter /note="synthetic bacterial promoter " primer_bind complement(3371..3399) /label=GFP-R /note="GFP, reverse primer. Does NOT anneal to EGFP" primer_bind 3994..4014 /label=GFP-F /note="GFP, forward primer. Does NOT anneal to EGFP" CDS 4037..4414 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MDAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRD DVTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWG REFALRDPAGNCVHFVAEEQD" polyA_signal 4544..4665 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4581..4600) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4635..4654 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4714..4730) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4714..4730) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4727..4749) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4738..4754 /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)." promoter complement(4762..4792) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4807..4828 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4945..4962) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5116..5704) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5196..5215) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5875..6735) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 6498..6517 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6736..6840) /gene="bla" /label=AmpR promoter primer_bind complement(6915..6934) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat 61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg 121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata 181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta 241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc 301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 361 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga 421 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg 481 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg 541 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact 601 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa 661 gctggctagc gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt 721 ggaattcagc caccatgggc agcagccatc atcatcatca tcacagcagc ggcctggtgc 781 cgcgcggcag ccatatgtca cgcaaaatcc gcgatttaat cgaatccaaa cgctttcaaa 841 acgtcatcac cgccattatt gtgctcaatg gcgctgtgct gggtctgctg accgatacaa 901 ccctgtcggc ctccagccaa aacctgctgg agcgtgtgga tcaactttgt ctgactatct 961 ttattgttga aatctccctg aaaatctacg cctatggcgt gcgcggcttt ttccgcagcg 1021 gctggaatct gtttgatttt gtgattgtgg ccatcgcgct tatgccggcc cagggtagcc 1081 tgtcggtgct gcgtaccttc cgtatcttcc gcgtcatgcg cctcgtatcg gtcatcccaa 1141 ccatgcgccg tgtggtgcaa ggcatgctct tggcactgcc gggcgtgggc tcggtagcgg 1201 cactgttgac ggtggtcttc tatattgcgg ctgtcatggc caccaatctc tacggggcaa 1261 ccttccctga atggtttggt gatcttagca agagcctgta cacactgttt caggtgatga 1321 ccttagagtc atggtctatg ggcattgtgc gtccagtgat gaacgttcat ccgaacgcat 1381 gggttttttt catcccgttc atcatgctca ccacctttac cgtgctcaac ctgtttattg 1441 gcattattgt agatgcaatg gcaatcacca aggaacagga ggaagaggcc aaaaccggtc 1501 accatcaaga acctatttct caaactcttc ttcatcttgg tgatcgtctt gatcgtattg 1561 aaaaacaact tgctcaaaat aatgaacttc ttcaacgtca acaacctcaa aaaaaataag 1621 gatccgcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg 1681 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 1741 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 1801 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 1861 gacaatagca gctttgcaaa gatggataaa gttttaaaca gagaggaatc tttgcagcta 1921 atggaccttc taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga 1981 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc 2041 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt 2101 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg 2161 caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct 2221 ctttacgggt tatggccctt gcgtgccttg aattacttcc acctggctgc agtacgtgat 2281 tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg 2341 agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg 2401 aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa 2461 tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca 2521 agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc 2581 ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg 2641 gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc 2701 gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct 2761 tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg 2821 tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc 2881 cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc 2941 gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga 3001 gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga 3061 gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt 3121 tcaggtgtcg tgaggaatta gcttggtact aatacgactc actataggga gacccaagct 3181 ggctaggtaa gctcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg 3241 gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata 3301 cgacaaggtg aggaactaaa ccatggcggt agaaaaaatg gctagcaaag gagaagaact 3361 tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt 3421 ttctgtcagt ggagagggtg aaggtgatgc tacatacgga aagcttaccc ttaaatttat 3481 ttgcactact ggaaaactac ctgttccatg gccaacactt gtcactactt tctcttatgg 3541 tgttcaatgc ttttcccgtt atccggatca tatgaaacgg catgactttt tcaagagtgc 3601 catgcccgaa ggttatgtac aggaacgcac tatatctttc aaagatgacg ggaactacaa 3661 gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt aatcgtatcg agttaaaagg 3721 tattgatttt aaagaagatg gaaacattct cggacacaaa ctcgagtaca actataactc 3781 acacaatgta tacatcacgg cagacaaaca aaagaatgga atcaaagcta acttcaaaat 3841 tcgtcacaac attgaagatg gatccgttca actagcagac cattatcaac aaaatactcc 3901 aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtcgacac aatctgccct 3961 ttcgaaagat cccaacgaaa agcgtgacca catggtcctt cttgagtttg taactgctgc 4021 tgggattaca catggcatgg atgccaagtt gaccagtgcc gttccggtgc tcaccgcgcg 4081 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt 4141 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca 4201 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct 4261 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat 4321 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa 4381 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac 4441 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat 4501 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc 4561 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 4621 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc 4681 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 4741 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 4801 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 4861 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 4921 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 4981 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 5041 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 5101 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 5161 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 5221 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 5281 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 5341 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 5401 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 5461 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 5521 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 5581 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 5641 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 5701 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 5761 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 5821 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 5881 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 5941 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 6001 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 6061 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 6121 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 6181 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 6241 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 6301 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 6361 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 6421 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 6481 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 6541 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 6601 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 6661 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 6721 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 6781 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 6841 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta 6901 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct 6961 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa 7021 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc 7081 gatgtacggg ccagatatac g //