LOCUS       Exported                7308 bp ds-DNA     circular SYN 06-AUG-2020
DEFINITION  His6-MBP-tev-G-Hs.KRAS4b(1-169) G13D.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7308)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Aug 12, 2020 from SnapGene Viewer 5.0.8
            https://www.snapgene.com
COMMENT     Entry Clone:   R989-E11, 3221 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..7308
                     /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..1790
                     /codon_start=1
                     /label=His6-MBP-tev-G-Hs.KRAS4b(1-169) G13D
                     /translation="MRSHHHHHHGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKV
                     TVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTW
                     DAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQE
                     PYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSI
                     AEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASP
                     NKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIM
                     PNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSGSDITSLYKKVGENLYFQGMT
                     EYKLVVVGAGDVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQ
                     EEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDL
                     PSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEK"
     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..1790
                     /codon_start=1
                     /gene="tev-GM-Hs.KRAS(2-169) G13D"
                     /label=tev-G-Hs.KRAS4b(1-169) G13D
                     /translation="ENLYFQGMTEYKLVVVGAGDVGKSALTIQLIQNHFVDEYDPTIED
                     SYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYR
                     EQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAF
                     YTLVREIRKHKEK"
     CDS             1260..1280
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(1789..1813)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1912..1929
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2095..2181
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2273..2300
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      2368..2415
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2452..2907
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(3022..3882)
                     /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(3883..3975)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4056..4644
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    4830..4972
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(5074..5265)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5837..6919)
                     /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(6920..6997)
                     /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 atgactgaat ataaacttgt ggtagttgga gctggtgacg
     1321 taggcaagag tgccttgacg atacagctaa ttcagaatca ttttgtggac gaatatgatc
     1381 caacaataga ggattcctac aggaagcaag tagtaattga tggagaaacc tgtctcttgg
     1441 atattctcga cacagcaggt caagaggagt acagtgcaat gagggaccag tacatgagga
     1501 ctggggaggg ctttctttgt gtatttgcca taaataatac taaatcattt gaagatattc
     1561 accattatag agaacaaatt aaaagagtta aggactctga agatgtacct atggtcctag
     1621 taggaaataa atgtgatttg ccttctagaa cagtagacac aaaacaggct caggacttag
     1681 caagaagtta tggaattcct tttattgaaa catcagcaaa gacaagacag ggtgttgatg
     1741 atgccttcta tacattagtt cgagaaattc gaaaacataa agaaaagtaa tcaactttct
     1801 tgtacaaagt ggtgatatcg ctagctagat agctagaagc ttgcggccgc acagctgtat
     1861 acacgtgcaa gccagccaga actcgctcct gaagacccag aggatctcga gcaccaccac
     1921 caccaccact aatgttaatt aagttgggcg ttcctaggct gataaaacag aatttgcctg
     1981 gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta
     2041 gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata
     2101 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     2161 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
     2221 ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc
     2281 atcctgacgg atggcctttt tgcgtttcta caaactcttt tgtttatttt tctaaataca
     2341 ttcaaatatg tatccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     2401 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     2461 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     2521 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2581 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     2641 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     2701 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     2761 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     2821 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2881 ttaacaaaat attaacgttt acaatttctg gcggcacgat ggcatgagat tatcaaaaag
     2941 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3001 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3061 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     3121 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     3181 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     3241 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     3301 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     3361 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     3421 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     3481 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     3541 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     3601 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     3661 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     3721 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3781 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3841 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaatc
     3901 atgattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     3961 agaaaaataa acaaataggt catgaccaaa atcccttaac gtgagttttc gttccactga
     4021 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4081 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4141 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4201 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4261 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     4321 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     4381 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     4441 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4501 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4561 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4621 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4681 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     4741 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     4801 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     4861 tgcggtattt cacaccgcat atatggtgca ctctcagtac aatctgctct gatgccgcat
     4921 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     4981 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5041 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5101 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     5161 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     5221 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     5281 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     5341 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     5401 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     5461 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     5521 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     5581 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     5641 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     5701 caacgacagg agcacgatca tgctagtcat gccccgcgcc caccggaagg agctgactgg
     5761 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     5821 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5881 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     5941 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     6001 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     6061 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     6121 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     6181 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     6241 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     6301 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     6361 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     6421 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     6481 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     6541 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     6601 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     6661 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     6721 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     6781 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     6841 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     6901 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     6961 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     7021 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     7081 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     7141 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     7201 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     7261 acgatgcgtc cggcgtagag gatcgagatc gatctcgatc ccgcgaaa
//
