LOCUS       sequence-313283-        7797 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli protein expression of His6-MBP-tev-Hs.NF1 DEF (1198-1530).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7797)
  AUTHORS   Yan W, Markegard E, Dharmaiah S, Urisman A, Drew M, Esposito D, 
            Scheffzek K, Nissley DV, McCormick F, Simanshu DK
  TITLE     Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and 
            Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR.
  JOURNAL   Cell Rep. 2020 Jul 21;32(3):107909. doi: 
            10.1016/j.celrep.2020.107909.
  PUBMED    32697994
REFERENCE   2  (bases 1 to 7797)
  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"; journalName: "Cell Rep.";
            date: "2020-07-21"; volume: "32(3):107909. doi"; pages: " 
            10.1016/j.celrep.2020.107909"
COMMENT     Sequence Label: R711-X04-566: His6-MBP-tev-Hs.NF1iso2 (1198-1530)
FEATURES             Location/Qualifiers
     source          1..7797
                     /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(2281..2305)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2365..2397
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             2404..2421
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2587..2673
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2765..2792
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2846..2864)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2860..2907
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2944..3399
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3031..3050)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3241..3262
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3514..4305,4306..4374))
                     /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     4137..4156
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4375..4467)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4548..5136
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5037..5056
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5290..5307
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5322..5461
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5387..5406)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5547..5569
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5563..5754)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6326..7408)
                     /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     7370..7389
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7409..7486)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7692..7711
                     /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 gagactgtgc tggccgacag attcgagaga ctggtggaac
     1321 tggtgaccat gatgggcgac cagggcgagc tgcctattgc catggccctg gccaacgtgg
     1381 tgccctgtag ccagtgggat gagctggcca gggtcctcgt cacactgttc gacagccggc
     1441 acctgctgta ccagctgctg tggaatatgt tcagcaaaga ggtggagctg gccgactcca
     1501 tgcagaccct gttcagaggc aactccctgg ccagcaagat catgaccttt tgcttcaagg
     1561 tgtacggcgc cacctacctg cagaagctgc tggaccccct gctgaggatc gtgatcacca
     1621 gcagcgactg gcagcacgtg agcttcgagg tggaccccac cagactggaa cccagcgaga
     1681 gcctggaaga gaaccagcgg aacctgctgc agatgaccga gaagttcttc cacgccatca
     1741 tcagcagcag ctccgagttc cccccccagc tgagaagcgt gtgccactgc ctgtaccagg
     1801 tggtgtccca gcggttcccc cagaatagca tcggcgccgt gggcagcgcc atgttcctgc
     1861 ggttcatcaa ccctgccatc gtgagccctt acgaggccgg catcctggat aagaagcccc
     1921 ctccccggat cgagaggggc ctgaagctga tgagcaagat tctgcagagc attgccaacc
     1981 acgtgctgtt taccaaggaa gagcacatgc ggcccttcaa cgacttcgtg aagagcaact
     2041 tcgacgccgc caggcggttc ttcctggata tcgccagcga ctgccctacc agcgacgccg
     2101 tgaaccacag cctgtccttc atcagcgacg gcaacgtcct ggccctgcat agactgctgt
     2161 ggaacaacca ggaaaagatc ggccagtacc tgagcagcaa ccgggaccac aaggctgtgg
     2221 gcaggcggcc cttcgataag atggccaccc tgctggccta tctgggccct cccgagcact
     2281 aatcaacttt cttgtacaaa gtggtgatat cgctagctag atagctagaa gcttgcggcc
     2341 gcacagctgt atacacgtgc aagccagcca gaactcgctc ctgaagaccc agaggatctc
     2401 gagcaccacc accaccacca ctaatgttaa ttaagttggg cgttcctagg ctgataaaac
     2461 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     2521 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     2581 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     2641 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
     2701 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt
     2761 aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt
     2821 tttctaaata cattcaaata tgtatccgct gagcaataac tagcataacc ccttggggcc
     2881 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     2941 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3001 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3061 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3121 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3181 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3241 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3301 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3361 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc tggcggcacg atggcatgag
     3421 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3481 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3541 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3601 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     3661 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3721 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3781 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3841 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3901 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3961 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4021 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4081 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4141 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4201 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4261 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4321 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4381 cctttttcaa tcatgattga agcatttatc agggttattg tctcatgagc ggatacatat
     4441 ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca aaatccctta acgtgagttt
     4501 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4561 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4621 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4681 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4741 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4801 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4861 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4921 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4981 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5041 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5101 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5161 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5221 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5281 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     5341 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     5401 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     5461 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5521 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5581 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     5641 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     5701 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     5761 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     5821 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     5881 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     5941 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     6001 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     6061 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     6121 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     6181 ccgggtcctc aacgacagga gcacgatcat gctagtcatg ccccgcgccc accggaagga
     6241 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     6301 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6361 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     6421 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     6481 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     6541 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     6601 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     6661 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     6721 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     6781 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     6841 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     6901 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     6961 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     7021 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     7081 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     7141 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     7201 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     7261 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     7321 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     7381 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     7441 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     7501 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     7561 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     7621 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     7681 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     7741 atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaa
//
