LOCUS       sequence-327136-        7590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7590)
  AUTHORS   Lakshman B, Messing S, Schmid EM, Clogston JD, Gillette WK, Esposito
            D, Kessing B, Fletcher DA, Nissley DV, McCormick F, Stephen AG, 
            Jean-Francois FL
  TITLE     Quantitative biophysical analysis defines key components modulating 
            recruitment of the GTPase KRAS to the plasma membrane.
  JOURNAL   J Biol Chem. 2019 Feb 8;294(6):2193-2207. doi: 
            10.1074/jbc.RA118.005669. Epub 2018 Dec 17.
  PUBMED    30559287
REFERENCE   2  (bases 1 to 7590)
  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: 
            "10.1074/jbc.RA118.005669"; journalName: "J Biol Chem"; date: 
            "2019-02-8- 8"; volume: "294"; issue: "6"; pages: "2193-2207"
COMMENT     Sequence Label: R725-X20-566: His6-MBP-tev-Avi-Hs.LGALS3
FEATURES             Location/Qualifiers
     source          1..7590
                     /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"
     CDS             1278..1322
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     protein_bind    complement(2074..2098)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2158..2190
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             2197..2214
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2380..2466
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2558..2585
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2639..2657)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2653..2700
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2737..3192
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2824..2843)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3034..3055
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3307..4098,4099..4167))
                     /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     3930..3949
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4168..4260)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4341..4929
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4830..4849
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5083..5100
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5115..5254
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5180..5199)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5340..5362
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5356..5547)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6119..7201)
                     /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     7163..7182
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7202..7279)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7485..7504
                     /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 ctgaacgaca tcttcgaagc ccagaagatt gagtggcatg
     1321 aaggggccga taacttcagc ctgcacgacg ccctttccgg gtctggcaac cccaacccgc
     1381 aaggctggcc tggagcttgg ggcaaccagc ctgcgggtgc tggaggttac ccgggtgcca
     1441 gctaccccgg agcttacccc ggccaagcac ctcccggcgc atatccggga caggctcctc
     1501 ctggcgcata cccaggagcc cctggagcct atcctggtgc acccgctcct ggagtgtacc
     1561 ctgggccgcc atcaggacca ggggcgtacc cgagctccgg acagccgtca gccactggag
     1621 cctaccccgc gactggacct tacggagccc cagcaggacc actgatcgtc ccgtacaact
     1681 tgcccttgcc gggtggagtg gtgccccgga tgctcattac catcctgggc accgtgaagc
     1741 cgaacgccaa ccggattgcc ctggattttc agcgcggcaa tgacgtggcc ttccatttca
     1801 acccgcggtt taacgagaac aatagaaggg tcatcgtgtg caacaccaag ctcgacaaca
     1861 actggggccg cgaagaacgc cagtccgtgt tcccgttcga gtccggaaag cccttcaaga
     1921 tccaggtcct ggtcgagccc gaccacttca aagtggccgt gaacgacgcg cacctcctgc
     1981 aatacaatca cagagtgaag aagctcaacg agatctcgaa gctggggatt tccggcgata
     2041 tcgacctgac ctccgcctcg tacaccatga tctaatcaac tttcttgtac aaagtggtga
     2101 tatcgctagc tagatagcta gaagcttgcg gccgcacagc tgtatacacg tgcaagccag
     2161 ccagaactcg ctcctgaaga cccagaggat ctcgagcacc accaccacca ccactaatgt
     2221 taattaagtt gggcgttcct aggctgataa aacagaattt gcctggcggc agtagcgcgg
     2281 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg
     2341 tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
     2401 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     2461 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     2521 ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc
     2581 ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc
     2641 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     2701 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     2761 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2821 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2881 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2941 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3001 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3061 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3121 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3181 cgtttacaat ttctggcggc acgatggcat gagattatca aaaaggatct tcacctagat
     3241 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3301 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3361 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3421 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     3481 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3541 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3601 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     3661 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3721 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     3781 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     3841 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     3901 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     3961 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4021 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4081 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4141 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatcatgat tgaagcattt
     4201 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     4261 taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4321 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4381 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4441 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4501 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4561 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4621 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4681 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4741 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4801 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4861 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4921 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4981 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     5041 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     5101 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     5161 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     5221 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     5281 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5341 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     5401 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     5461 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5521 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5581 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5641 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5701 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5761 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5821 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5881 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5941 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     6001 catgctagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     6061 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     6121 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6181 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     6241 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     6301 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     6361 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
     6421 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     6481 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     6541 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     6601 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     6661 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     6721 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     6781 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     6841 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     6901 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     6961 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     7021 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     7081 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     7141 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     7201 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     7261 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     7321 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     7381 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     7441 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     7501 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     7561 aggatcgaga tcgatctcga tcccgcgaaa
//
