LOCUS       Exported                7350 bp ds-DNA     circular SYN 07-AUG-2020
DEFINITION  His6-MBP-tev-Avi-KRAS4b (1-169) G12V.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7350)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Aug 12, 2020 from SnapGene Viewer 5.0.8
            https://www.snapgene.com
COMMENT     Entry Clone:   R701-E55, 3263 bps DNA circular (attL1, attL2 sites)
            Destination Vector:   pDest-566, 9367 bps DNA circular (attR1, attR2
            sites)
            Recombination using LR clonase to produce Expression Clone
FEATURES             Location/Qualifiers
     source          1..7350
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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             90..1832
                     /codon_start=1
                     /label=His6-MBP-tev-Avi-Hs.KRAS4b(2-169) G12V
                     /translation="MRSHHHHHHGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKV
                     TVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTW
                     DAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQE
                     PYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSI
                     AEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASP
                     NKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIM
                     PNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSGSDITSLYKKVGENLYFQGLN
                     DIFEAQKIEWHEGTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVID
                     GETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDS
                     EDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRK
                     HKEK"
     CDS             99..116
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             120..1217
                     /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="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1074..1083
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    1236..1260
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1260..1833
                     /codon_start=1
                     /gene="tev-Avi-KRAS4b (1-169) G12V"
                     /label=tev-Avi-KRAS4b(2-169) G12V
                     /translation="ENLYFQGLNDIFEAQKIEWHEGTEYKLVVVGAVGVGKSALTIQLI
                     QNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFA
                     INNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFI
                     ETSAKTRQGVDDAFYTLVREIRKHKEK"
     CDS             1260..1280
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1278..1322
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     protein_bind    complement(1831..1855)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1954..1971
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2137..2223
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2315..2342
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      2410..2457
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2494..2949
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(3064..3924)
                     /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"
     promoter        complement(3925..4017)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4098..4686
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    4872..5014
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(5116..5307)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5879..6961)
                     /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"
     promoter        complement(6962..7039)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
       61 ttgtttaact ttaagaagga gatatacata tgagatccca tcaccatcac catcacggca
      121 aaatcgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg
      181 ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg
      241 ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct
      301 tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc
      361 cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg
      421 gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc
      481 tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag
      541 cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga
      601 ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg
      661 tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa
      721 acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg
      781 aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga
      841 attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg
      901 tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg
      961 aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg
     1021 ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacca
     1081 tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt
     1141 atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc
     1201 tgaaagacgc gcagactaat tcgggatctg atatcacaag tttgtacaaa aaagttggcg
     1261 aaaacctgta cttccaaggc ctgaacgaca tcttcgaggc tcagaaaatc gaatggcacg
     1321 aaggcactga atataaactt gtggtagttg gagctgttgg cgtaggcaag agtgccttga
     1381 cgatacagct aattcagaat cattttgtgg acgaatatga tccaacaata gaggattcct
     1441 acaggaagca agtagtaatt gatggagaaa cctgtctctt ggatattctc gacacagcag
     1501 gtcaagagga gtacagtgca atgagggacc agtacatgag gactggggag ggctttcttt
     1561 gtgtatttgc cataaataat actaaatcat ttgaagatat tcaccattat agagaacaaa
     1621 ttaaaagagt taaggactct gaagatgtac ctatggtcct agtaggaaat aaatgtgatt
     1681 tgccttctag aacagtagac acaaaacagg ctcaggactt agcaagaagt tatggaattc
     1741 cttttattga aacatcagca aagacaagac agggtgttga tgatgccttc tatacattag
     1801 ttcgagaaat tcgaaaacat aaagaaaagt aatcaacttt cttgtacaaa gtggtgatat
     1861 cgctagctag atagctagaa gcttgcggcc gcacagctgt atacacgtgc aagccagcca
     1921 gaactcgctc ctgaagaccc agaggatctc gagcaccacc accaccacca ctaatgttaa
     1981 ttaagttggg cgttcctagg ctgataaaac agaatttgcc tggcggcagt agcgcggtgg
     2041 tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg
     2101 ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg
     2161 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     2221 aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga
     2281 cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt
     2341 tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata tgtatccgct
     2401 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     2461 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     2521 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2581 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2641 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2701 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2761 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2821 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     2881 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     2941 ttacaatttc tggcggcacg atggcatgag attatcaaaa aggatcttca cctagatcct
     3001 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3061 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3121 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3181 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     3241 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3301 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3361 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3421 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3481 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3541 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3601 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     3661 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     3721 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3781 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3841 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3901 gacacggaaa tgttgaatac tcatactctt cctttttcaa tcatgattga agcatttatc
     3961 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4021 gtcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4081 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4141 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4201 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4261 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4321 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4381 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4441 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4501 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4561 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4621 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4681 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4741 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4801 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4861 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4921 atatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4981 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     5041 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5101 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     5161 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     5221 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5281 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5341 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5401 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5461 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5521 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5581 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5641 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5701 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5761 catgctagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     5821 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     5881 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5941 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     6001 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     6061 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     6121 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
     6181 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     6241 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     6301 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     6361 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     6421 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     6481 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     6541 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     6601 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     6661 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     6721 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     6781 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     6841 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     6901 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     6961 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     7021 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     7081 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     7141 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     7201 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     7261 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     7321 aggatcgaga tcgatctcga tcccgcgaaa
//
