LOCUS       sequence-356797-        5764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 10*His tagged human BTLA intracellular domain 
            (AA190-289, Y243F, 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, Y243F, Y257F,
            ...
FEATURES             Location/Qualifiers
     source          1..5764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      68..523
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(155..174)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     365..386
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(615..1430)
                     /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(615..1430)
                     /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     1338..1357
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1422..1441
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1552..2140
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2041..2060
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2294..2311
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2326..2465
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2391..2410)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2551..2573
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2567..2758)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3567..4649)
                     /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     4611..4630
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4650..4727)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4933..4952
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5036..5055
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5036..5054
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5055..5079
                     /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             5094..5116
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5135..5164
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5174..5191
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5195..5227
                     /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             5540..5563
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5600..5623
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5664..5681
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5734..5752)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(5748..5764,1..31)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
       61 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
      121 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      181 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      241 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
      301 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
      361 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
      421 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
      481 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
      541 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      601 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
      661 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
      721 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
      781 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
      841 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
      901 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
      961 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     1021 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     1081 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     1141 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     1201 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     1261 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     1321 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     1381 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     1441 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga
     1501 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     1561 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     1621 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     1681 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     1741 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     1801 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     1861 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     1921 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     1981 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     2041 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2101 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     2161 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     2221 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     2281 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     2341 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     2401 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     2461 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     2521 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     2581 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     2641 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     2701 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     2761 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     2821 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     2881 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     2941 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     3001 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     3061 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     3121 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     3181 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     3241 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     3301 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     3361 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     3421 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     3481 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     3541 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3601 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     3661 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     3721 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     3781 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     3841 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     3901 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     3961 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     4021 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     4081 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     4141 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     4201 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     4261 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     4321 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     4381 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     4441 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     4501 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     4561 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     4621 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     4681 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     4741 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     4801 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     4861 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     4921 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     4981 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     5041 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     5101 aactttaaga aggagatata ccatgggcag cagccatcat catcatcatc atcatcacca
     5161 tcacagcagc ggcctggtgc cgcgcggcag ccatatggct agcatgactg gtggacagca
     5221 aatgggtcgc ggagacacag caggaaggga aattaacctg gttgatgctc accttaagag
     5281 tgagcaaaca gaagcaagca ccaggcaaaa ttcccaagta ctgctatcag aaactggaat
     5341 ttatgataat gaccctgacc tttgtttcag gatgcaggaa gggtctgaag ttttttctaa
     5401 tccatgcctg gaagaaaaca aaccaggcat tgtttttgct tccctgaacc attctgtcat
     5461 tggactgaac tcaagactgg caagaaatgt aaaagaagca ccaacagaat ttgcatccat
     5521 atgtgtgagg agtggatcct ggagccaccc gcagttcgag aaaggtggag gttccggagg
     5581 tggatcggga ggtggatcgt ggagccaccc gcagttcgaa aaataattcg agctccgtcg
     5641 acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca
     5701 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     5761 ttgg
//
