LOCUS       Exported                7407 bp ds-DNA     circular SYN 07-AUG-2020
DEFINITION  His6-MBP-tev-Avi-KRAS4b Q61L.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7407)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Aug 12, 2020 from SnapGene Viewer 5.0.8
            https://www.snapgene.com
COMMENT     Entry Clone:   R701-E23, 3320 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..7407
                     /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..1889
                     /codon_start=1
                     /label=His6-MBP-tev-Avi-KRAS4b(2-188) Q61L
                     /translation="MRSHHHHHHGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKV
                     TVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTW
                     DAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQE
                     PYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSI
                     AEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASP
                     NKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIM
                     PNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSGSDITSLYKKVGENLYFQGLN
                     DIFEAQKIEWHEGTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVID
                     GETCLLDILDTAGLEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDS
                     EDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRK
                     HKEKMSKDGKKKKKKSKTKCVIM"
     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..1889
                     /codon_start=1
                     /label=tev-Avi-KRAS4b(2-188) Q61L
                     /translation="ENLYFQGLNDIFEAQKIEWHEGTEYKLVVVGAGGVGKSALTIQLI
                     QNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGLEEYSAMRDQYMRTGEGFLCVFA
                     INNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFI
                     ETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSKTKCVIM"
     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(1888..1912)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2011..2028
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2194..2280
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2372..2399
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      2467..2514
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2551..3006
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(3121..3981)
                     /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(3982..4074)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4155..4743
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    4929..5071
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(5173..5364)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5936..7018)
                     /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(7019..7096)
                     /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 gagctggtgg cgtaggcaag agtgccttga
     1381 cgatacagct aattcagaat cattttgtgg acgaatatga tccaacaata gaggattcct
     1441 acaggaagca agtagtaatt gatggagaaa cctgtctctt ggatattctc gacacagcag
     1501 gtctagagga 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 aaagaaaaga tgagcaaaga tggtaaaaag aagaaaaaga
     1861 agtcaaagac aaagtgtgta attatgtaat caactttctt gtacaaagtg gtgatatcgc
     1921 tagctagata gctagaagct tgcggccgca cagctgtata cacgtgcaag ccagccagaa
     1981 ctcgctcctg aagacccaga ggatctcgag caccaccacc accaccacta atgttaatta
     2041 agttgggcgt tcctaggctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc
     2101 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
     2161 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     2221 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     2281 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     2341 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
     2401 gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctgag
     2461 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     2521 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     2581 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     2641 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2701 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2761 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     2821 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2881 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2941 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     3001 caatttctgg cggcacgatg gcatgagatt atcaaaaagg atcttcacct agatcctttt
     3061 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     3121 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     3181 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     3241 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     3301 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     3361 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     3421 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     3481 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     3541 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     3601 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     3661 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     3721 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     3781 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     3841 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     3901 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     3961 acggaaatgt tgaatactca tactcttcct ttttcaatca tgattgaagc atttatcagg
     4021 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaataggtc
     4081 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4141 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4201 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4261 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     4321 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4381 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4441 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4501 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4561 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4621 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4681 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4741 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4801 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4861 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4921 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4981 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     5041 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     5101 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5161 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     5221 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     5281 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     5341 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     5401 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     5461 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     5521 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     5581 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     5641 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     5701 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     5761 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     5821 gctagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     5881 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     5941 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6001 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     6061 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     6121 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     6181 agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg
     6241 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     6301 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     6361 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     6421 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     6481 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     6541 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     6601 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     6661 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     6721 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     6781 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     6841 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     6901 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     6961 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     7021 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     7081 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     7141 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     7201 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     7261 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     7321 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     7381 atcgagatcg atctcgatcc cgcgaaa
//
