LOCUS       sequence-322427-        6620 bp    DNA     circular SYN 14-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6620)
  AUTHORS   Morcos P, Thapar N, Tusneem N, Stacey D, Tamanoi F
  TITLE     Identification of neurofibromin mutants that exhibit allele 
            specificity or increased Ras affinity resulting in suppression of 
            activated ras alleles.
  JOURNAL   Mol Cell Biol. 1996 May . 16(5):2496-503.
  PUBMED    8628317
REFERENCE   2  (bases 1 to 6620)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell 
            Biol. 1996 May . 16(5):2496-503."
COMMENT     Sequence Label: pGBT9-NF1-GRD
FEATURES             Location/Qualifiers
     source          1..6620
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             425..865
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     terminator      2009..2196
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2064..2088)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2249..2923)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(2924..3025)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(3039..3055)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3039..3055)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3052..3074)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3063..3079
                     /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)."
     promoter        complement(join(3087..3093,3094..3111,3112..3117))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3132..3153
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3270..3287)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3441..4029)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3521..3540)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4200..4991,4992..5060))
                     /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     4823..4842
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5061..5165)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5233..5251
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5289..5311)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5411..5430
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      5447..6611
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        join(6616..6620,1..397)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
ORIGIN
        1 caacttcttt tctttttttt tcttttctct ctcccccgtt gttgtctcac catatccgca
       61 atgacaaaaa aatgatggaa gacactaaag gaaaaaatta acgacaaaga cagcaccaac
      121 agatgtcgtt gttccagagc tgatgagggg tatctcgaag cacacgaaac tttttccttc
      181 cttcattcac gcacactact ctctaatgag caacggtata cggccttcct tccagttact
      241 tgaatttgaa ataaaaaaaa gtttgctgtc ttgctatcaa gtataaatag acctgcaatt
      301 attaatcttt tgtttcctcg tcattgttct cgttcccttt cttccttgtt tctttttctg
      361 cacaatattt caagctatac caagcataca atcaactcca agcttgaagc aagcctcctg
      421 aaagatgaag ctactgtctt ctatcgaaca agcatgcgat atttgccgac ttaaaaagct
      481 caagtgctcc aaagaaaaac cgaagtgcgc caagtgtctg aagaacaact gggagtgtcg
      541 ctactctccc aaaaccaaaa ggtctccgct gactagggca catctgacag aagtggaatc
      601 aaggctagaa agactggaac agctatttct actgattttt cctcgagaag accttgacat
      661 gattttgaaa atggattctt tacaggatat aaaagcattg ttaacaggat tatttgtaca
      721 agataatgtg aataaagatg ccgtcacaga tagattggct tcagtggaga ctgatatgcc
      781 tctaacattg agacagcata gaataagtgc gacatcatca tcggaagaga gtagtaacaa
      841 aggtcaaaga cagttgactg tatcgccgga attgggtacc gatctccaga caagagctac
      901 atttatggaa gttctgacaa aaatccttca acaaggcaca gaatttgaca tacttgcaga
      961 aacagtattg gctgatcggt ttgagagatt ggtggaactg gtcacaatga tgggtgatca
     1021 aggagaactc cctatagcga tggctctggc caatgtggtt ccttgttctc agtgggatga
     1081 actagctcga gttctggtta ctctgtttga ttctcggcat ttactctacc aactgctctg
     1141 gaacatgttt tctaaagaag tagaattggc agactccatg cagactctct tccgaggcaa
     1201 cagcttggcc agtaaaataa tgacattctg tttcaaggta tatggtgcta cctatctaca
     1261 aaaactcctg gatcctttat tacgaattgt gatcacatcc tctgattggc aacatgttag
     1321 ctttgaagtg gatcctacca ggttagaacc atcagagagc cttgaggaaa accagcggaa
     1381 cctccttcag atgactgaaa agttcttcca tgccatcatc agttcctcct cagaattccc
     1441 ccctcaactt cgaagtgtgt gccactgttt ataccaggtg gttagccagc gtttccctca
     1501 gaacagcatc ggtgcagtag gaagtgccat gttcctcaga tttatcaatc ctgccattgt
     1561 ctcaccgtat gaagcaggga ttttagataa aaagccacca cctagaatcg aaaggggctt
     1621 gaagttaatg tcaaagatac ttcagagtat tgccaatcat gttctcttca caaaagaaga
     1681 acatatgcgg cctttcaatg attttgtgaa aagcaacttt gatgcagcac gcaggttttt
     1741 ccttgatata gcatctgatt gtcctacaag tgatgcagta aatcatagtc tttccttcat
     1801 aagtgacggc aatgtgcttg ctttacatcg tctactctgg aacaatcagg agaaaattgg
     1861 gcagtatctt tccagcaaca gggatcataa agctgttgga agacgacctt ttgataagat
     1921 ggcaacactt cttgcatacc tgggtcctcc agagcacaaa cctgtggcag atacacactc
     1981 agtcgacctg cagccaagct aattccgggc gaatttctta tgatttatga tttttattat
     2041 taaataagtt ataaaaaaaa taagtgtata caaattttaa agtgactctt aggttttaaa
     2101 acgaaaattc ttattcttga gtaactcttt cctgtaggtc aggttgcttt ctcaggtata
     2161 gcatgaggtc gctcttattg accacacctc taccggcatg ccggcaagtg cacaaacaat
     2221 acttaaataa atactactca gtaataacct atttcttagc atttttgacg aaatttgcta
     2281 ttttgttaga gtcttttaca ccatttgtct ccacacctcc gcttacatca acaccaataa
     2341 cgccatttaa tctaagcgca tcaccaacat tttctggcgt cagtccacca gctaacataa
     2401 aatgtaagct ttcggggctc tcttgccttc caacccagtc agaaatcgag ttccaatcca
     2461 aaagttcacc tgtcccacct gcttctgaat caaacaaggg aataaacgaa tgaggtttct
     2521 gtgaagctgc actgagtagt atgttgcagt cttttggaaa tacgagtctt ttaataactg
     2581 gcaaaccgag gaactcttgg tattcttgcc acgactcatc tccatgcagt tggacgatat
     2641 caatgccgta atcattgacc agagccaaaa catcctcctt aggttgatta cgaaacacgc
     2701 caaccaagta tttcggagtg cctgaactat ttttatatgc ttttacaaga cttgaaattt
     2761 tccttgcaat aaccgggtca attgttctct ttctattggg cacacatata atacccagca
     2821 agtcagcatc ggaatctaga gcacattctg cggcctctgt gctctgcaag ccgcaaactt
     2881 tcaccaatgg accagaacta cctgtgaaat taataacaga catactccaa gctgcctttg
     2941 tgtgcttaat cacgtatact cacgtgctca atagtcacca atgccctccc tcttggccct
     3001 ctccttttct tttttcgacc gaattaattc gtaatcatgt catagctgtt tcctgtgtga
     3061 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     3121 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     3181 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3241 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3301 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3361 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3421 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     3481 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     3541 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3601 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     3661 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3721 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3781 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3841 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     3901 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3961 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4021 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4081 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4141 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4201 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4261 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4321 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4381 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4441 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4501 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     4561 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4621 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     4681 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     4741 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     4801 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     4861 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     4921 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     4981 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5041 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5101 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5161 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     5221 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     5281 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     5341 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     5401 cttaactatg cggcatcaga gcagattgta ctgagagtgc accataacgc atttaagcat
     5461 aaacacgcac tatgccgttc ttctcatgta tatatatata caggcaacac gcagatatag
     5521 gtgcgacgtg aacagtgagc tgtatgtgcg cagctcgcgt tgcattttcg gaagcgctcg
     5581 ttttcggaaa cgctttgaag ttcctattcc gaagttccta ttctctagct agaaagtata
     5641 ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa gacgcacttt caaaaaacca
     5701 aaaacgcacc ggactgtaac gagctactaa aatattgcga ataccgcttc cacaaacatt
     5761 gctcaaaagt atctctttgc tatatatctc tgtgctatat ccctatataa cctacccatc
     5821 cacctttcgc tccttgaact tgcatctaaa ctcgacctct acatttttta tgtttatctc
     5881 tagtattact ctttagacaa aaaaattgta gtaagaacta ttcatagagt gaatcgaaaa
     5941 caatacgaaa atgtaaacat ttcctatacg tagtatatag agacaaaata gaagaaaccg
     6001 ttcataattt tctgaccaat gaagaatcat caacgctatc actttctgtt cacaaagtat
     6061 gcgcaatcca catcggtata gaatataatc ggggatgcct ttatcttgaa aaaatgcacc
     6121 cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt caggcttttt ttatggaaga
     6181 gaaaatagac accaaagtag ccttcttcta accttaacgg acctacagtg caaaaagtta
     6241 tcaagagact gcattataga gcgcacaaag gagaaaaaaa gtaatctaag atgctttgtt
     6301 agaaaaatag cgctctcggg atgcattttt gtagaacaaa aaagaagtat agattctttg
     6361 ttggtaaaat agcgctctcg cgttgcattt ctgttctgta aaaatgcagc tcagattctt
     6421 tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt acaaaaatga agcacagatt
     6481 cttcgttggt aaaatagcgc tttcgcgttg catttctgtt ctgtaaaaat gcagctcaga
     6541 ttctttgttt gaaaaattag cgctctcgcg ttgcattttt gttctacaaa atgaagcaca
     6601 gatgcttcgt tgcttgcatg
//
