LOCUS       sequence-313162-        7782 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli protein expression of His6-MBP-tev-Hs.NF1iso2 (1203-1530).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7782)
  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 7782)
  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-X12-566: His6-MBP-tev-Hs.NF1iso2 (1203-1530)
FEATURES             Location/Qualifiers
     source          1..7782
                     /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(2266..2290)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2350..2382
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             2389..2406
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2572..2658
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2750..2777
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2831..2849)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2845..2892
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2929..3384
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3016..3035)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3226..3247
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3499..4290,4291..4359))
                     /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     4122..4141
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4360..4452)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4533..5121
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5022..5041
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5275..5292
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5307..5446
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5372..5391)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5532..5554
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5548..5739)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6311..7393)
                     /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     7355..7374
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7394..7471)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7677..7696
                     /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 gacagattcg agagactggt ggaactggtg accatgatgg
     1321 gcgaccaggg cgagctgcct attgccatgg ccctggccaa cgtggtgccc tgtagccagt
     1381 gggatgagct ggccagggtc ctcgtcacac tgttcgacag ccggcacctg ctgtaccagc
     1441 tgctgtggaa tatgttcagc aaagaggtgg agctggccga ctccatgcag accctgttca
     1501 gaggcaactc cctggccagc aagatcatga ccttttgctt caaggtgtac ggcgccacct
     1561 acctgcagaa gctgctggac cccctgctga ggatcgtgat caccagcagc gactggcagc
     1621 acgtgagctt cgaggtggac cccaccagac tggaacccag cgagagcctg gaagagaacc
     1681 agcggaacct gctgcagatg accgagaagt tcttccacgc catcatcagc agcagctccg
     1741 agttcccccc ccagctgaga agcgtgtgcc actgcctgta ccaggtggtg tcccagcggt
     1801 tcccccagaa tagcatcggc gccgtgggca gcgccatgtt cctgcggttc atcaaccctg
     1861 ccatcgtgag cccttacgag gccggcatcc tggataagaa gccccctccc cggatcgaga
     1921 ggggcctgaa gctgatgagc aagattctgc agagcattgc caaccacgtg ctgtttacca
     1981 aggaagagca catgcggccc ttcaacgact tcgtgaagag caacttcgac gccgccaggc
     2041 ggttcttcct ggatatcgcc agcgactgcc ctaccagcga cgccgtgaac cacagcctgt
     2101 ccttcatcag cgacggcaac gtcctggccc tgcatagact gctgtggaac aaccaggaaa
     2161 agatcggcca gtacctgagc agcaaccggg accacaaggc tgtgggcagg cggcccttcg
     2221 ataagatggc caccctgctg gcctatctgg gccctcccga gcactaatca actttcttgt
     2281 acaaagtggt gatatcgcta gctagatagc tagaagcttg cggccgcaca gctgtataca
     2341 cgtgcaagcc agccagaact cgctcctgaa gacccagagg atctcgagca ccaccaccac
     2401 caccactaat gttaattaag ttgggcgttc ctaggctgat aaaacagaat ttgcctggcg
     2461 gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg
     2521 ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa
     2581 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     2641 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
     2701 gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc
     2761 ctgacggatg gcctttttgc gtttctacaa actcttttgt ttatttttct aaatacattc
     2821 aaatatgtat ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     2881 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     2941 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3001 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3061 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3121 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3181 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3241 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3301 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     3361 acaaaatatt aacgtttaca atttctggcg gcacgatggc atgagattat caaaaaggat
     3421 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3481 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3541 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3601 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3661 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3721 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3781 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3841 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3901 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3961 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4021 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4081 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4141 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4201 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4261 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4321 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatcatg
     4381 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4441 aaaataaaca aataggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4501 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4561 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4621 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     4681 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4741 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4801 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4861 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4921 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4981 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5041 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5101 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     5161 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     5221 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     5281 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     5341 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5401 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     5461 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5521 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5581 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     5641 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     5701 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     5761 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     5821 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     5881 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     5941 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     6001 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     6061 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     6121 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     6181 caggagcacg atcatgctag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     6241 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     6301 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     6361 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     6421 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     6481 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     6541 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
     6601 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     6661 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     6721 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     6781 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     6841 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     6901 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     6961 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     7021 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     7081 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     7141 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     7201 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     7261 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     7321 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     7381 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     7441 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     7501 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     7561 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     7621 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     7681 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     7741 cgtccggcgt agaggatcga gatcgatctc gatcccgcga aa
//
