LOCUS       sequence-313760-        6141 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli protein expression of His6-tev-Hs.CALM1(2-148).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6141)
  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 6141)
  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-X65-527: His6-tev-Hs.CALM1(2-148)
FEATURES             Location/Qualifiers
     source          1..6141
                     /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"
     CDS             180..626
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTE
                     AELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAE
                     LRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     protein_bind    complement(625..649)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             709..741
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             748..765
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      931..1017
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1109..1136
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1190..1208)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1204..1251
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1288..1743
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1375..1394)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1585..1606
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1858..2649,2650..2718))
                     /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     2481..2500
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2719..2811)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2892..3480
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3381..3400
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3634..3651
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3666..3805
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3731..3750)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3891..3913
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3907..4098)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4670..5752)
                     /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     5714..5733
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5753..5830)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6036..6055
                     /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 ttccaaggcg
      181 ctgatcagct gaccgaagaa cagattgctg aattcaagga agccttctcc ctatttgata
      241 aagatggcga tggcaccatc acaacaaagg aacttggaac tgtcatgagg tcactgggtc
      301 agaacccaac agaagctgaa ttgcaggata tgatcaatga agtggatgct gatggtaatg
      361 gcaccattga cttccccgaa tttttgacca tgatggctag aaaaatgaaa gatacagata
      421 gtgaagaaga aatccgtgag gcattccgag tctttgacaa ggatggcaat ggttatatca
      481 gtgcagcaga actacgtcac gtcatgacaa acttaggaga aaaactaaca gatgaagaag
      541 tagatgaaat gatcagagaa gcagatattg atggagacgg acaagtcaac tatgaagaat
      601 tcgtacagat gatgactgca aaataatcaa ctttcttgta caaagtggtg atatcgctag
      661 ctagatagct agaagcttgc ggccgcacag ctgtatacac gtgcaagcca gccagaactc
      721 gctcctgaag acccagagga tctcgagcac caccaccacc accactaatg ttaattaagt
      781 tgggcgttcc taggctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac
      841 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc
      901 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
      961 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
     1021 ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg
     1081 ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg
     1141 tttctacaaa ctcttttgtt tatttttcta aatacattca aatatgtatc cgctgagcaa
     1201 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     1261 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     1321 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1381 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1441 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1501 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1561 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1621 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1681 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     1741 tttctggcgg cacgatggca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     1801 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     1861 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     1921 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     1981 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     2041 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     2101 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     2161 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     2221 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     2281 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     2341 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     2401 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     2461 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     2521 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     2581 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     2641 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     2701 gaaatgttga atactcatac tcttcctttt tcaatcatga ttgaagcatt tatcagggtt
     2761 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggtcatg
     2821 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2881 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2941 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3001 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3061 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3121 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3181 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3241 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3301 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3361 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3421 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3481 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3541 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3601 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3661 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     3721 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     3781 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     3841 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     3901 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     3961 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     4021 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     4081 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     4141 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     4201 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     4261 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     4321 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     4381 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     4441 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     4501 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgctagt
     4561 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     4621 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     4681 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4741 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     4801 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     4861 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     4921 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg
     4981 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     5041 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     5101 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     5161 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     5221 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     5281 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     5341 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     5401 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     5461 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     5521 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     5581 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     5641 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     5701 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     5761 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     5821 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     5881 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     5941 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     6001 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     6061 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     6121 atcgatctcg atcccgcgaa a
//
