LOCUS       sequence-356794-        5764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 10*His tagged human BTLA intracellular domain 
            (AA190-289, Y226F, Y243F, Y282F)-TwinStrep.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5764)
  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 5764)
  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: pET28-His10-BTLAicd (AA190-289, Y226F, Y243F,
            ...
FEATURES             Location/Qualifiers
     source          1..5764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      52..507
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(139..158)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     349..370
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(599..1414)
                     /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(599..1414)
                     /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     1322..1341
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1406..1425
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1536..2124
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2025..2044
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2278..2295
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2310..2449
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2375..2394)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2535..2557
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2551..2742)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3551..4633)
                     /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     4595..4614
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4634..4711)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4917..4936
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5020..5039
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5020..5038
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5039..5063
                     /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             5078..5100
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5119..5148
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5158..5175
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5179..5211
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             5524..5547
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5584..5607
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5648..5665
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5718..5736)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(5732..5764,1..15)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
       61 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      121 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
      181 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
      241 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
      301 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
      361 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
      421 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
      481 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
      541 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
      601 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
      661 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
      721 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
      781 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
      841 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
      901 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
      961 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     1021 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     1081 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     1141 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     1201 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     1261 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     1321 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     1381 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     1441 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     1501 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     1561 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     1621 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1681 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     1741 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     1801 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     1861 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     1921 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     1981 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2041 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2101 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2161 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     2221 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     2281 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     2341 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     2401 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     2461 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     2521 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     2581 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     2641 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     2701 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     2761 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     2821 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     2881 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     2941 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     3001 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     3061 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     3121 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     3181 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     3241 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     3301 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     3361 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     3421 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     3481 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     3541 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3601 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     3661 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     3721 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     3781 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     3841 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     3901 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     3961 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4021 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4081 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4141 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     4201 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     4261 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     4321 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     4381 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     4441 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     4501 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     4561 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     4621 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     4681 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     4741 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     4801 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     4861 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     4921 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     4981 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
     5041 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
     5101 tataccatgg gcagcagcca tcatcatcat catcatcatc accatcacag cagcggcctg
     5161 gtgccgcgcg gcagccatat ggctagcatg actggtggac agcaaatggg tcgcggagac
     5221 acagcaggaa gggaaattaa cctggttgat gctcacctta agagtgagca aacagaagca
     5281 agcaccaggc aaaattccca agtactgcta tcagaaactg gaatttttga taatgaccct
     5341 gacctttgtt tcaggatgca ggaagggtct gaagtttttt ctaatccatg cctggaagaa
     5401 aacaaaccag gcattgttta tgcttccctg aaccattctg tcattggact gaactcaaga
     5461 ctggcaagaa atgtaaaaga agcaccaaca gaatttgcat ccatatgtgt gaggagtgga
     5521 tcctggagcc acccgcagtt cgagaaaggt ggaggttccg gaggtggatc gggaggtgga
     5581 tcctggagcc acccgcagtt cgaaaaataa ttcgagctcc gtcgacaagc ttgcggccgc
     5641 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     5701 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     5761 ggtc
//
