LOCUS       sequence-313785-        5934 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli protein expression of His6-tev-Hs.CALM1(1-80).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5934)
  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 5934)
  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-X71-527: His6-tev-Hs.CALM1(1-80)
FEATURES             Location/Qualifiers
     source          1..5934
                     /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(418..442)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             502..534
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             541..558
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      724..810
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      902..929
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(983..1001)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      997..1044
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1081..1536
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1168..1187)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1378..1399
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1651..2442,2443..2511))
                     /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     2274..2293
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2512..2604)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2685..3273
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3174..3193
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3427..3444
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3459..3598
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3524..3543)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3684..3706
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3700..3891)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4463..5545)
                     /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     5507..5526
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5546..5623)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5829..5848
                     /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 gatacataat
      421 caactttctt gtacaaagtg gtgatatcgc tagctagata gctagaagct tgcggccgca
      481 cagctgtata cacgtgcaag ccagccagaa ctcgctcctg aagacccaga ggatctcgag
      541 caccaccacc accaccacta atgttaatta agttgggcgt tcctaggctg ataaaacaga
      601 atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga
      661 aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg
      721 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
      781 tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag
      841 caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag
      901 cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctttt gtttattttt
      961 ctaaatacat tcaaatatgt atccgctgag caataactag cataacccct tggggcctct
     1021 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     1081 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1141 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1201 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1261 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1321 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1381 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1441 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1501 acgcgaattt taacaaaata ttaacgttta caatttctgg cggcacgatg gcatgagatt
     1561 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     1621 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     1681 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     1741 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     1801 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     1861 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     1921 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     1981 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     2041 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     2101 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     2161 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     2221 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     2281 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     2341 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     2401 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     2461 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     2521 ttttcaatca tgattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     2581 aatgtattta gaaaaataaa caaataggtc atgaccaaaa tcccttaacg tgagttttcg
     2641 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2701 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2761 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2821 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2881 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2941 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3001 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3061 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3121 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3181 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3241 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3301 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3361 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3421 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3481 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     3541 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     3601 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     3661 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     3721 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     3781 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     3841 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     3901 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     3961 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4021 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4081 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     4141 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     4201 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     4261 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     4321 ggtcctcaac gacaggagca cgatcatgct agtcatgccc cgcgcccacc ggaaggagct
     4381 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4441 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4501 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     4561 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     4621 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     4681 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     4741 agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     4801 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     4861 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     4921 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     4981 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     5041 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     5101 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     5161 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     5221 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     5281 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     5341 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     5401 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     5461 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     5521 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     5581 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     5641 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     5701 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     5761 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     5821 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     5881 ccggccacga tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaa
//
