LOCUS       sequence-356783-        6346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GST fused human BTLA intracellular domain (AA190-289, 
            Y257F, Y282F)-TwinStrep.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6346)
  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 6346)
  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: pET28a-GST-BTLAicd (AA190-289, Y257F,
            Y282F)-T...
FEATURES             Location/Qualifiers
     source          1..6346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     955..974
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(994..1071)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1277..1296
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1380..1399
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1380..1398
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1399..1423
                     /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)."
     RBS             1438..1460
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1467..2120
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             2127..2150
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2466..2489
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2526..2549
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2590..2607
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2660..2678)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2674..2721
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2758..3213
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2845..2864)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3055..3076
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3305..4120)
                     /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(3305..4120)
                     /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     4028..4047
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4112..4131
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4242..4830
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4731..4750
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4984..5001
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5016..5155
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5081..5100)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5241..5263
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5257..5448)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6257..6346,1..993))
                     /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"
ORIGIN
        1 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
       61 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
      121 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
      181 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
      241 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
      301 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
      361 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
      421 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
      481 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
      541 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
      601 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
      661 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
      721 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
      781 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
      841 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
      901 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
      961 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     1021 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     1081 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     1141 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     1201 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     1261 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     1321 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
     1381 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
     1441 gtttaacttt aagaaggaga tataccatgt cccctatact aggttattgg aaaattaagg
     1501 gccttgtgca acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt
     1561 tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt
     1621 ttcccaatct tccttattat attgatggtg atgttaaatt aacacagtct atggccatca
     1681 tacgttatat agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga
     1741 tttcaatgct tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata
     1801 gtaaagactt tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa
     1861 tgttcgaaga tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg
     1921 acttcatgtt gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg
     1981 cgttcccaaa attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt
     2041 acttgaaatc cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg
     2101 gtggcgacca tcctccaaaa tcggatctgg aagttctgtt ccaggggccc ctgggatccg
     2161 acacagcagg aagggaaatt aacctggttg atgctcacct taagagtgag caaacagaag
     2221 caagcaccag gcaaaattcc caagtactgc tatcagaaac tggaatttat gataatgacc
     2281 ctgacctttg tttcaggatg caggaagggt ctgaagttta ttctaatcca tgcctggaag
     2341 aaaacaaacc aggcattgtt tttgcttccc tgaaccattc tgtcattgga ctgaactcaa
     2401 gactggcaag aaatgtaaaa gaagcaccaa cagaatttgc atccatatgt gtgaggagtg
     2461 gatcctggag ccacccgcag ttcgagaaag gtggaggttc cggaggtgga tcgggaggtg
     2521 gatcgtggag ccacccgcag ttcgaaaaat aattcgagct ccgtcgacaa gcttgcggcc
     2581 gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     2641 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     2701 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     2761 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2821 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2881 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2941 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     3001 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3061 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3121 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3181 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     3241 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     3301 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     3361 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     3421 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     3481 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     3541 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     3601 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     3661 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     3721 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     3781 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     3841 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     3901 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     3961 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     4021 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     4081 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     4141 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     4201 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4261 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4321 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4381 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4441 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4501 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4561 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4621 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4681 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4741 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4801 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4861 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4921 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4981 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     5041 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     5101 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     5161 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     5221 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     5281 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     5341 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     5401 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     5461 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     5521 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     5581 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     5641 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     5701 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     5761 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     5821 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     5881 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     5941 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     6001 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     6061 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     6121 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     6181 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     6241 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6301 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attggg
//
