LOCUS       sequence-146459-        5625 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construct for expressing Grb2 in E. Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5625)
  AUTHORS   Su X, Ditlev JA, Hui E, Xing W, Banjade S, Okrut J, King DS, Taunton
            J, Rosen MK, Vale RD
  TITLE     Phase separation of signaling molecules promotes T cell receptor 
            signal transduction.
  JOURNAL   Science. 2016 Apr 29;352(6285):595-9. doi: 10.1126/science.aad9964. 
            Epub 2016 Apr 7.
  PUBMED    27056844
REFERENCE   2  (bases 1 to 5625)
  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: 
            "10.1126/science.aad9964"; journalName: "Science"; date: 
            "2016-04-29- 29"; volume: "352"; issue: "6285"; pages: "595-9"
COMMENT     Sequence Label: GST-PS-Grb2 (XSB363)
FEATURES             Location/Qualifiers
     source          1..5625
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1675..1697)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1842..1860)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1928..2032
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2033..2101,2102..2893)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2251..2270)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3064..3652
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3553..3572
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3806..3823
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3896..3973
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3974..5056
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3993..4012)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5069..5090
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5105..5110,5111..5128,5129..5135)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5143..5159
                     /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)."
     primer_bind     5167..5183
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5167..5183
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5179..5625,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5199..5216)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5199..5215)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5208..5230)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc atggaagcca
      961 tcgccaaata tgacttcaaa gctactgcag acgacgagct gagcttcaaa aggggggaca
     1021 tcctcaaggt tttgaacgaa gaatgtgatc agaactggta caaggcagag cttaatggaa
     1081 aagacggctt cattcccaag aactacatag aaatgaaacc acatccgtgg ttttttggca
     1141 aaatccccag agccaaggca gaagaaatgc ttagcaaaca gcggcacgat ggggcctttc
     1201 ttatccgaga gagtgagagc gctcctgggg acttctccct ctctgtcaag tttggaaacg
     1261 atgtgcagca cttcaaggtg ctccgagatg gagccgggaa gtacttcctc tgggtggtga
     1321 agttcaattc tttgaatgag ctggtggatt atcacagatc tacatctgtc tccagaaacc
     1381 agcagatatt cctgcgggac atagaacagg tgccacagca gccgacatac gtccaggccc
     1441 tctttgactt tgatccccag gaggatggag agctgggctt ccgccgggga gattttatcc
     1501 atgtcatgga taactcagac cccaactggt ggaaaggagc ttgccacggg cagaccggca
     1561 tgtttccccg caattatgtc acccccgtga accggaacgt ctaagtcgac tcgagcggcc
     1621 gcatcgtgac tgactgacga tctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     1681 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     1741 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     1801 acgtagcgat agcggagtgt ataattcttg aagacgaaag ggcctcgtga tacgcctatt
     1861 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     1921 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1981 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     2041 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     2101 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     2161 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     2221 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga
     2281 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     2341 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     2401 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     2461 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     2521 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
     2581 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     2641 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     2701 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     2761 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     2821 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     2881 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     2941 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     3001 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3061 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3121 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3181 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     3241 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3301 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3361 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3421 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3481 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3541 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3601 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3661 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3721 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3781 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     3841 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcataa attccgacac
     3901 catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     3961 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     4021 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     4081 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     4141 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     4201 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     4261 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     4321 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     4381 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     4441 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     4501 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     4561 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     4621 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     4681 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     4741 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac
     4801 cgaagacagc tcatgttata tcccgccgtc aaccaccatc aaacaggatt ttcgcctgct
     4861 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     4921 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     4981 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     5041 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     5101 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     5161 ttcacacagg aaacagctat gaccatgatt acggattcac tggccgtcgt tttacaacgt
     5221 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     5281 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     5341 ctgaatggcg aatggcgctt tgcctggttt ccggcaccag aagcggtgcc ggaaagctgg
     5401 ctggagtgcg atcttcctga ggccgatact gtcgtcgtcc cctcaaactg gcagatgcac
     5461 ggttacgatg cgcccatcta caccaacgta acctatccca ttacggtcaa tccgccgttt
     5521 gttcccacgg agaatccgac gggttgttac tcgctcacat ttaatgttga tgaaagctgg
     5581 ctacaggaag gccagacgcg aattattttt gatggcgttg gaatt
//
