LOCUS       sequence-356705-        5264 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GST fused human PD-1 intracellular domain (AA194-288, 
            Y223F, Y248F).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5264)
  AUTHORS   Xu X, Hou B, Fulzele A, Masubuchi T, Zhao Y, Wu Z, Hu Y, Jiang Y, Ma
            Y, Wang H, Bennett EJ, Fu G, Hui E
  TITLE     PD-1 and BTLA regulate T cell signaling differentially and only 
            partially through SHP1 and SHP2.
  JOURNAL   J Cell Biol. 2020 Jun 1;219(6). pii: 151801. doi: 
            10.1083/jcb.201905085.
  PUBMED    32437509
REFERENCE   2  (bases 1 to 5264)
  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: "J Cell 
            Biol."; date: "2020-06-1"; volume: "219(6). pii: 151801. doi"; 
            pages: " 10.1083/jcb.201905085"
COMMENT     Sequence Label: pGEX-6p-2-GST-PD-1icd (AA194-288, Y223F, Y248F)
FEATURES             Location/Qualifiers
     source          1..5264
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(223..228,229..244,245..251)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    259..275
                     /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             298..951
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     909..931
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             958..981
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1354..1376)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1521..1539)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1607..1711
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1712..1780,1781..2572)
                     /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(1930..1949)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2743..3331
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3232..3251
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3485..3502
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3575..3652
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3653..4735
                     /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(3672..3691)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4784..4789,4790..4807,4808..4814)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4822..4838
                     /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     4846..4862
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4846..4862
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4858..5264,1..121)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4878..4895)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4878..4894)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4887..4909)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ggaaggccag acgcgaatta tttttgatgg cgttggaatt agcttatcga ctgcacggtg
       61 caccaatgct tctggcgtca ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta
      121 aatcactgca taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg
      181 ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatcgg
      241 ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac agtattcatg
      301 tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg
      361 gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg
      421 cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt
      481 gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg
      541 ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat
      601 attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat
      661 tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca
      721 tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt
      781 gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa
      841 cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg
      901 cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa atcggatctg
      961 gaagttctgt tccaggggcc cctgggatcc cgggccgcac gagggacaat aggagccagg
     1021 cgcaccggcc agcccctgaa ggaggacccc tcagccgtgc ctgtgttctc tgtggacttt
     1081 ggggagctgg atttccagtg gcgagagaag accccggagc cccccgtgcc ctgtgtccct
     1141 gagcagacgg agtttgccac cattgtcttt cctagcggaa tgggcacctc atcccccgcc
     1201 cgcaggggct cagccgacgg ccctcggagt gcccagccac tgaggcctga ggatggacac
     1261 tgctcttggc ccctgtaacc cgggtcgact cgagcggccg catcgtgact gactgacgat
     1321 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     1381 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     1441 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     1501 taattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     1561 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1621 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     1681 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     1741 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     1801 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     1861 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     1921 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     1981 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     2041 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     2101 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     2161 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     2221 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     2281 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     2341 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     2401 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     2461 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     2521 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     2581 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     2641 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     2701 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2761 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2821 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2881 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2941 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3001 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3061 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3121 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3181 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3241 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3301 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3361 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3421 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3481 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3541 gcatctgtgc ggtatttcac accgcataaa ttccgacacc atcgaatggc gcaaaacctt
     3601 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     3661 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     3721 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     3781 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     3841 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     3901 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     3961 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     4021 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     4081 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     4141 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     4201 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     4261 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     4321 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     4381 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     4441 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     4501 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     4561 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     4621 ggtgaaaaga aaaaccacgc tagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     4681 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     4741 acgcaattaa tgtaagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     4801 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     4861 accatgatta cggattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     4921 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     4981 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcttt
     5041 gcctggtttc cggcaccaga agcggtgccg gaaagctggc tggagtgcga tcttcctgag
     5101 gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg gttacgatgc gcccatctac
     5161 accaacgtga cctatcccat tacggtcaat ccgccgtttg ttcccacgga gaatccgacg
     5221 ggttgttact cgctcacatt taatgttgat gaaagctggc taca
//
