LOCUS       sequence-488800-        7305 bp    DNA     circular SYN 08-OCT-2025
DEFINITION  Bacterial protein expression of His6-MBP-tev-Hs.KRAS4b(1-169) G13V.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7305)
  TITLE     Esposito Ras Family Bacterial Expression Plasmids
REFERENCE   2  (bases 1 to 7305)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 8, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: R919-X77-566
FEATURES             Location/Qualifiers
     source          1..7305
                     /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             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
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1191..1214
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     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..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             1873..1905
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     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"
     primer_bind     complement(2354..2372)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     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"
     primer_bind     complement(2539..2558)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2749..2770
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3022..3813,3814..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"
     primer_bind     3645..3664
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     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"
     primer_bind     4545..4564
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4798..4815
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4830..4969
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4895..4914)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5055..5077
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5071..5262)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5834..6916)
                     /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     6878..6897
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6917..6994)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7200..7219
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
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 gctggtgtcg
     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 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4921 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     4981 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5041 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5101 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     5161 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     5221 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     5281 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     5341 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     5401 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     5461 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     5521 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     5581 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     5641 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     5701 cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     5761 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     5821 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5881 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     5941 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     6001 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     6061 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg
     6121 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     6181 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     6241 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     6301 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     6361 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     6421 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     6481 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     6541 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     6601 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     6661 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     6721 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     6781 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     6841 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     6901 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     6961 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     7021 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     7081 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     7141 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     7201 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     7261 atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg cgaaa
//
