LOCUS       sequence-488963-        6712 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses C2 and GAP domains of human p120RasGAP (residues 581-1047 
            wild type) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6712)
  AUTHORS   Paul ME, Vish KJ, Boggon TJ
  TITLE     Michaelis-Menten kinetics of RasGAP proteins by a rapid 
            fluorescence-based assay.
  JOURNAL   Methods. 2025 Dec;244:108-117. doi: 10.1016/j.ymeth.2025.09.003. 
            Epub 2025 Sep 8.
  PUBMED    40930400
REFERENCE   2  (bases 1 to 6712)
  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: "Methods."; 
            date: "2025-12"; volume: "244:108-117. doi"; pages: " 
            10.1016/j.ymeth.2025.09.003. Epub 2025 Sep 8"
COMMENT     Sequence Label: pET-p120RasGAP-581-1047-WT
FEATURES             Location/Qualifiers
     source          1..6712
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /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     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3489..3508
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3489..3507
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3508..3532
                     /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)."
     CDS             3575..3592
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3614..3634
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5045..5068
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(5131..5149)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5145..5192
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5229..5684
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5316..5335)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5526..5547
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5776..6591)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5776..6591)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6499..6518
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6583..6602
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc
     3481 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     3541 tttaaattaa ctttaagaag gagatataca tatgcatcac caccatcacc atggatcgct
     3601 ggttccgaga tctgaaaacc tgtattttca gggatccagt ccagggacat ccaataaacg
     3661 ccttcgtcag gtcagcagcc ttgttttaca tattgaagaa gcccataaac tcccagtaaa
     3721 acattttact aatccatatt gtaacatcta cctgaatagt gtccaagtag caaaaactca
     3781 tgcaagggaa gggcaaaacc cagtatggtc agaagagttt gtctttgatg atcttcctcc
     3841 tgacatcaat agatttgaaa taactcttag taataaaaca aagaaaagca aagatcctga
     3901 tatcttattt atgcgctgcc agttgagccg attacagaaa gggcatgcca cagatgaatg
     3961 gtttctgctc agctcccata taccattaaa aggtattgaa ccagggtccc tgcgtgttcg
     4021 agcacgatac tctatggaaa aaatcatgcc agaagaagag tacagtgaat ttaaagagct
     4081 tatactgcaa aaggaacttc atgtagtcta tgctttatca catgtatgtg gacaagaccg
     4141 aacactactg gccagcatcc tactgaggat ttttcttcac gaaaagcttg aatcgttgtt
     4201 gttatgcaca ctaaatgaca gagaaataag catggaagat gaagccacta ccctatttcg
     4261 agccacaaca cttgcaagca ccttgatgga gcagtatatg aaagccactg ctacacagtt
     4321 tgttcatcat gctttgaaag actctatttt aaagataatg gaaagcaagc agtcttgtga
     4381 gttaagtcca tcaaagttag aaaaaaatga agatgtgaac actaatttaa cacacctatt
     4441 gaacatactt tcagagcttg tggagaaaat attcatggct tcagaaatac ttccaccgac
     4501 attgagatat atttatgggt gtttacagaa atctgttcag cataagtggc ctacaaatac
     4561 caccatgaga acaagagttg ttagtggttt tgtttttctt cgactcatct gtcctgccat
     4621 cctgaatcca cggatgttca atatcatctc agattctcca agtcctattg ctgcaagaac
     4681 actgatatta gtggctaaat ctgtgcagaa cttagcaaat cttgtggaat ttggagctaa
     4741 ggagccctac atggaaggtg tcaatccatt catcaaaagc aacaaacatc gtatgatcat
     4801 gtttttagat gaacttggga atgtacctga acttccggac actacagagc attctagaac
     4861 ggacctgtcc cgtgatttag cagcattgca tgagatttgc gtggctcatt cagatgaact
     4921 tcgaacgctc agtaatgagc gtggtgcaca gcagcacgta ttgaaaaagc ttctggctat
     4981 aacagaactg cttcaacaaa aacaaaacca gtatacaaaa accaatgatg tcaggtagct
     5041 cgagcaccac caccaccacc accaccacta attgattaat acctaggctg ctaaacaaag
     5101 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     5161 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     5221 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     5281 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     5341 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     5401 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     5461 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5521 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5581 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5641 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     5701 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5761 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     5821 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     5881 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     5941 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     6001 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     6061 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     6121 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     6181 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     6241 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     6301 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     6361 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     6421 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     6481 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     6541 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     6601 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     6661 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tc
//
