LOCUS       sequence-218047-        5817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construct for expressing Gads SH3, SH2, and SH3 domain in E. Coli, 
            with a C-terminal Cys for labeling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5817)
  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 5817)
  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-Gads 1-155 GB1 Gads 261-330-Cys (XSB365)
FEATURES             Location/Qualifiers
     source          1..5817
                     /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"
     CDS             1419..1580
                     /codon_start=1
                     /product="B1 domain of Streptococcal protein G   "
                     /label=GB1
                     /note="effective as a solubilizing fusion partner (Cheng 
                     and Patel, 2004)"
                     /translation="YKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
                     ATKTFTVTE"
     primer_bind     complement(1867..1889)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2034..2052)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2120..2224
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2225..2293,2294..3085)
                     /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(2443..2462)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3256..3844
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3745..3764
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3998..4015
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4088..4165
                     /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             4166..5248
                     /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(4185..4204)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5261..5282
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5297..5302,5303..5320,5321..5327)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5335..5351
                     /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     5359..5375
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5359..5375
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5371..5817,1..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(5391..5408)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5391..5407)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5400..5422)
                     /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 atggaagctg
      961 ttgccaagtt tgatttcact gcttcaggtg aggatgaact gagctttcac actggagatg
     1021 ttttgaagat tttaagtaac caagaggagt ggtttaaggc ggagcttggg agccaggaag
     1081 gatatgtgcc caagaatttc atagacatcc agtttcccaa atggtttcac gaaggcctct
     1141 ctcgacacca ggcagagaac ttactcatgg gcaaggaggt tggcttcttc atcatccggg
     1201 ccagccagag ctccccaggg gacttctcca tctctgtcag gcatgaggat gacgttcaac
     1261 acttcaaggt catgcgagac aacaagggta attactttct gtggactgag aagtttccat
     1321 ccctaaataa gctggtagac tactacagga caaattccat ctccagacag aagcagatct
     1381 tccttagaga cagaacccga gaagaccagg gtcacgagta caagcttatc ctgaacggta
     1441 aaaccctgaa aggtgaaacc accaccgaag ctgttgacgc tgctaccgcg gaaaaagttt
     1501 tcaaacagta cgctaacgac aacggtgttg acggtgaatg gacctacgac gacgctacca
     1561 aaaccttcac ggtaaccgaa cacacagacc cagtgcagct ccaggcggca gggcgagtgc
     1621 ggtgggcccg ggcgctgtat gactttgagg ccctggagga tgacgagctg gggttccaca
     1681 gcggggaggt ggtggaggtc ctggatagct ccaacccatc ctggtggacc ggccgcctgc
     1741 acaacaagct gggcctcttc cctgccaact acgtggcacc catgacccgg tgctaagtcg
     1801 actcgagcgg ccgcatcgtg actgactgac gatctgcctc gcgcgtttcg gtgatgacgg
     1861 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     1921 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     1981 catgacccag tcacgtagcg atagcggagt gtataattct tgaagacgaa agggcctcgt
     2041 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     2101 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2161 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2221 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     2281 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     2341 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     2401 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     2461 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     2521 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     2581 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     2641 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     2701 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     2761 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     2821 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     2881 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     2941 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     3001 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     3061 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     3121 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     3181 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3241 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3301 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3361 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     3421 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3481 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3541 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3601 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3661 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3721 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3781 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3841 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3901 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3961 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     4021 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     4081 aaattccgac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga
     4141 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     4201 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     4261 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     4321 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     4381 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     4441 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     4501 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     4561 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     4621 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     4681 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     4741 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     4801 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     4861 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     4921 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
     4981 atacgacgat accgaagaca gctcatgtta tatcccgccg tcaaccacca tcaaacagga
     5041 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     5101 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     5161 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     5221 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     5281 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     5341 gcggataaca atttcacaca ggaaacagct atgaccatga ttacggattc actggccgtc
     5401 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     5461 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     5521 cagttgcgca gcctgaatgg cgaatggcgc tttgcctggt ttccggcacc agaagcggtg
     5581 ccggaaagct ggctggagtg cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac
     5641 tggcagatgc acggttacga tgcgcccatc tacaccaacg taacctatcc cattacggtc
     5701 aatccgccgt ttgttcccac ggagaatccg acgggttgtt actcgctcac atttaatgtt
     5761 gatgaaagct ggctacagga aggccagacg cgaattattt ttgatggcgt tggaatt
//
