LOCUS       sequence-313757-        5913 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli protein expression of His6-tev-Hs.CALM1(76-148).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5913)
  AUTHORS   Agamasu C, Ghirlando R, Taylor T, Messing S, Tran TH, Bindu L, 
            Tonelli M, Nissley DV, McCormick F, Stephen AG
  TITLE     KRAS Prenylation Is Required for Bivalent Binding with Calmodulin in
            a Nucleotide-Independent Manner.
  JOURNAL   Biophys J. 2019 Mar 19;116(6):1049-1063. doi: 
            10.1016/j.bpj.2019.02.004. Epub 2019 Feb 15.
  PUBMED    30846362
REFERENCE   2  (bases 1 to 5913)
  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.bpj.2019.02.004"; journalName: "Biophys J"; date: 
            "2019-03-19- 19"; volume: "116"; issue: "6"; pages: "1049-1063"
COMMENT     Sequence Label: R701-X72-527: His6-tev-Hs.CALM1(76-148)
FEATURES             Location/Qualifiers
     source          1..5913
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..21
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2..20
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    21..45
                     /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             60..82
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             105..122
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    135..159
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             159..179
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(397..421)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             481..513
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             520..537
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      703..789
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      881..908
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(962..980)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      976..1023
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1060..1515
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1147..1166)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1357..1378
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1630..2421,2422..2490))
                     /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     2253..2272
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2491..2583)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2664..3252
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3153..3172
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3406..3423
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3438..3577
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3503..3522)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3663..3685
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3679..3870)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4442..5524)
                     /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     5486..5505
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5525..5602)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5808..5827
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
       61 ttgtttaact ttaagaagga gatatacata tgagatctgg atcccaccat caccatcacc
      121 atagatctga tatcacaagt ttgtacaaaa aagttggcga aaacctgtac ttccaaggca
      181 tgaaagatac agatagtgaa gaagaaatcc gtgaggcatt ccgagtcttt gacaaggatg
      241 gcaatggtta tatcagtgca gcagaactac gtcacgtcat gacaaactta ggagaaaaac
      301 taacagatga agaagtagat gaaatgatca gagaagcaga tattgatgga gacggacaag
      361 tcaactatga agaattcgta cagatgatga ctgcataatc aactttcttg tacaaagtgg
      421 tgatatcgct agctagatag ctagaagctt gcggccgcac agctgtatac acgtgcaagc
      481 cagccagaac tcgctcctga agacccagag gatctcgagc accaccacca ccaccactaa
      541 tgttaattaa gttgggcgtt cctaggctga taaaacagaa tttgcctggc ggcagtagcg
      601 cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta
      661 gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      721 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      781 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
      841 gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat
      901 ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta
      961 tccgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1021 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     1081 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1141 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1201 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1261 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1321 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1381 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1441 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1501 taacgtttac aatttctggc ggcacgatgg catgagatta tcaaaaagga tcttcaccta
     1561 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     1621 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     1681 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     1741 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     1801 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     1861 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     1921 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     1981 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     2041 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     2101 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     2161 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     2221 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     2281 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     2341 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     2401 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     2461 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatcat gattgaagca
     2521 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     2581 aaataggtca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2641 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2701 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2761 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     2821 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2881 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2941 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3001 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3061 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3121 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3181 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3241 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3301 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3361 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3421 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3481 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     3541 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     3601 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3661 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     3721 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     3781 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     3841 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     3901 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     3961 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     4021 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     4081 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     4141 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     4201 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     4261 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     4321 gatcatgcta gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     4381 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     4441 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4501 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     4561 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     4621 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     4681 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
     4741 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     4801 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     4861 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     4921 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     4981 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     5041 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     5101 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     5161 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     5221 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     5281 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     5341 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     5401 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     5461 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     5521 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     5581 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     5641 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     5701 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     5761 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     5821 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     5881 tagaggatcg agatcgatct cgatcccgcg aaa
//
