LOCUS       sequence-353464-        6218 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Baculo expression of GST tag fused to SNAPtag and human Shp2 tandem 
            SH2 domains.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6218)
  AUTHORS   Hui E, Cheung J, Zhu J, Su X, Taylor MJ, Wallweber HA, Sasmal DK, 
            Huang J, Kim JM, Mellman I, Vale RD
  TITLE     T cell costimulatory receptor CD28 is a primary target for 
            PD-1-mediated inhibition.
  JOURNAL   Science. 2017 Mar 31;355(6332):1428-1433. doi: 
            10.1126/science.aaf1292. Epub 2017 Mar 9.
  PUBMED    28280247
REFERENCE   2  (bases 1 to 6218)
  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.aaf1292"; journalName: "Science"; date: 
            "2017-03-31- 31"; volume: "355"; issue: "6332"; pages: "1428-1433"
COMMENT     Sequence Label: pGEX6P2(GST-SNAP-Shp2tSH2)
FEATURES             Location/Qualifiers
     source          1..6218
                     /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             948..1493
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     primer_bind     complement(2268..2290)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2435..2453)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2521..2625
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2626..2694,2695..3486)
                     /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(2844..2863)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3657..4245
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4146..4165
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4399..4416
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4489..4566
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4567..5649
                     /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     complement(4586..4605)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5698..5703,5704..5721,5722..5728)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5736..5752
                     /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     5760..5776
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5760..5776
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5772..6218,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5792..5809)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5792..5808)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5801..5823)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga 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 cctgggtatg gacaaagatt
      961 gcgaaatgaa acgtaccacc ctggatagcc cgctgggcaa actggaactg agcggctgcg
     1021 aacagggcct gcatgaaatt aaactgctgg gtaaaggcac cagcgcggcc gatgcggttg
     1081 aagttccggc cccggccgcc gtgctgggtg gtccggaacc gctgatgcag gcgaccgcgt
     1141 ggctgaacgc gtattttcat cagccggaag cgattgaaga atttccggtt ccggcgctgc
     1201 atcatccggt gtttcagcag gagagcttta cccgtcaggt gctgtggaaa ctgctgaaag
     1261 tggttaaatt tggcgaagtg attagctatc agcagctggc ggccctggcg ggtaatccgg
     1321 cggccaccgc cgccgttaaa accgcgctga gcggtaaccc ggtgccgatt ctgattccgt
     1381 gccatcgtgt ggttagctct agcggtgcgg ttggcggtta tgaaggtggt ctggcggtga
     1441 aagagtggct gctggcccat gaaggtcatc gtctgggtaa accgggtctg ggaggtagcg
     1501 gtagcggtag cggtggaggt ggaagcagcg gtagatggtt tcacccaaat atcactggtg
     1561 tggaggcaga aaacctactg ttgacaagag gagttgatgg cagttttttg gcaaggccta
     1621 gtaaaagtaa ccctggagac ttcacacttt ccgttagaag aaatggagct gtcacccaca
     1681 tcaagattca gaacactggt gattactatg acctgtatgg aggggagaaa tttgccactt
     1741 tggctgagtt ggtccagtat tacatggaac atcacgggca attaaaagag aagaatggag
     1801 atgtcattga gcttaaatat cctctgaact gtgcagatcc tacctctgaa aggtggtttc
     1861 atggacatct ctctgggaaa gaagcagaga aattattaac tgaaaaagga aaacatggta
     1921 gttttcttgt acgagagagc cagagccacc ctggagattt tgttctttct gtgcgcactg
     1981 gtgatgacaa aggggagagc aatgacggca agtctaaagt gacccatgtt atgattcgct
     2041 gtcaggaact gaaatacgac gttggtggag gagaacggtt tgattctttg acagatcttg
     2101 tggaacatta taagaagaat cctatggtgg aaacattggg tacagtacta caactcaagc
     2161 agccccttaa cacgtaagga tccccaggaa ttcccgggtc gactcgagcg gccgcatcgt
     2221 gactgactga cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     2281 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     2341 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     2401 gatagcggag tgtataattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     2461 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     2521 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     2581 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     2641 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     2701 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     2761 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     2821 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     2881 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     2941 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3001 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3061 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3121 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
     3181 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3241 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3301 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     3361 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3421 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     3481 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     3541 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     3601 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3661 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3721 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3781 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     3841 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3901 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3961 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4021 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4081 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4141 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4201 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4261 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4321 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4381 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     4441 tattttctcc ttacgcatct gtgcggtatt tcacaccgca taaattccga caccatcgaa
     4501 tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
     4561 gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag
     4621 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
     4681 gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
     4741 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
     4801 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
     4861 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
     4921 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
     4981 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
     5041 attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
     5101 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
     5161 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
     5221 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
     5281 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
     5341 gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac
     5401 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
     5461 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
     5521 ttgcccgtct cactggtgaa aagaaaaacc acgctagcgc ccaatacgca aaccgcctct
     5581 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     5641 gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cccaggcttt
     5701 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     5761 aggaaacagc tatgaccatg attacggatt cactggccgt cgttttacaa cgtcgtgact
     5821 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     5881 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     5941 gcgaatggcg ctttgcctgg tttccggcac cagaagcggt gccggaaagc tggctggagt
     6001 gcgatcttcc tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg cacggttacg
     6061 atgcgcccat ctacaccaac gtgacctatc ccattacggt caatccgccg tttgttccca
     6121 cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc tggctacagg
     6181 aaggccagac gcgaattatt tttgatggcg ttggaatt
//
