LOCUS       sequence-356795-        5764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 10*His tagged human BTLA intracellular domain 
            (AA190-289, Y226F, Y257F, 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, Y257F,
            ...
FEATURES             Location/Qualifiers
     source          1..5764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      73..528
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(160..179)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     370..391
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(620..1435)
                     /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(620..1435)
                     /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     1343..1362
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1427..1446
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1557..2145
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2046..2065
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2299..2316
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2331..2470
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2396..2415)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2556..2578
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2572..2763)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3572..4654)
                     /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     4616..4635
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4655..4732)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4938..4957
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5041..5060
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5041..5059
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5060..5084
                     /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             5099..5121
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5140..5169
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5179..5196
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5200..5232
                     /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             5545..5568
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5605..5628
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5669..5686
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5739..5757)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(5753..5764,1..36)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
       61 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
      121 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
      181 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      241 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
      301 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
      361 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
      421 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
      481 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
      541 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      601 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
      661 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
      721 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
      781 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
      841 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
      901 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
      961 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     1021 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     1081 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1141 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1201 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1261 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1321 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     1381 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     1441 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     1501 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1561 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1621 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1681 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1741 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1801 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1861 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1921 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1981 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     2041 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2101 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2161 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2221 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2281 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     2341 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     2401 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     2461 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     2521 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     2581 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     2641 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     2701 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     2761 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     2821 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     2881 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     2941 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     3001 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     3061 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     3121 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     3181 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     3241 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     3301 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     3361 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     3421 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     3481 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     3541 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3601 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3661 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     3721 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     3781 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     3841 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     3901 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     3961 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     4021 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     4081 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     4141 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     4201 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     4261 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     4321 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     4381 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     4441 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     4501 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     4561 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     4621 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     4681 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     4741 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     4801 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     4861 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     4921 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     4981 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     5041 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     5101 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcatcat
     5161 caccatcaca gcagcggcct ggtgccgcgc ggcagccata tggctagcat gactggtgga
     5221 cagcaaatgg gtcgcggaga cacagcagga agggaaatta acctggttga tgctcacctt
     5281 aagagtgagc aaacagaagc aagcaccagg caaaattccc aagtactgct atcagaaact
     5341 ggaatttttg ataatgaccc tgacctttgt ttcaggatgc aggaagggtc tgaagtttat
     5401 tctaatccat gcctggaaga aaacaaacca ggcattgttt ttgcttccct gaaccattct
     5461 gtcattggac tgaactcaag actggcaaga aatgtaaaag aagcaccaac agaatttgca
     5521 tccatatgtg tgaggagtgg atcctggagc cacccgcagt tcgagaaagg tggaggttcc
     5581 ggaggtggat cgggaggtgg atcgtggagc cacccgcagt tcgaaaaata attcgagctc
     5641 cgtcgacaag cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc
     5701 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     5761 accc
//
