LOCUS       sequence-356793-        5764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 10*His tagged human BTLA intracellular domain 
            (AA190-289, Y226F, Y243F, Y257F)-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      47..502
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(134..153)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     344..365
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(594..1409)
                     /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(594..1409)
                     /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     1317..1336
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1401..1420
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1531..2119
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2020..2039
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2273..2290
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2305..2444
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2370..2389)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2530..2552
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2546..2737)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3546..4628)
                     /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     4590..4609
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4629..4706)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4912..4931
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5015..5034
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5015..5033
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5034..5058
                     /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             5073..5095
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5114..5143
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5153..5170
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5174..5206
                     /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             5519..5542
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5579..5602
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5643..5660
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5713..5731)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(5727..5764,1..10)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
       61 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
      121 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
      181 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
      241 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
      301 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
      361 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
      421 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
      481 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
      541 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
      601 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
      661 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
      721 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
      781 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
      841 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
      901 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
      961 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     1021 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     1081 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     1141 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     1201 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     1261 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     1321 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     1381 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     1441 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     1501 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     1561 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     1621 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     1681 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     1741 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     1801 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     1861 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     1921 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     1981 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     2041 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     2101 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     2161 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     2221 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     2281 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     2341 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     2401 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     2461 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     2521 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     2581 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     2641 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     2701 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     2761 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     2821 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     2881 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     2941 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     3001 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     3061 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     3121 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     3181 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     3241 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     3301 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     3361 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     3421 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     3481 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     3541 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3601 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     3661 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     3721 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     3781 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     3841 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     3901 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     3961 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     4021 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     4081 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     4141 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     4201 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     4261 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     4321 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     4381 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     4441 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     4501 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     4561 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     4621 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     4681 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     4741 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     4801 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     4861 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     4921 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     4981 ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg
     5041 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     5101 catgggcagc agccatcatc atcatcatca tcatcaccat cacagcagcg gcctggtgcc
     5161 gcgcggcagc catatggcta gcatgactgg tggacagcaa atgggtcgcg gagacacagc
     5221 aggaagggaa attaacctgg ttgatgctca ccttaagagt gagcaaacag aagcaagcac
     5281 caggcaaaat tcccaagtac tgctatcaga aactggaatt tttgataatg accctgacct
     5341 ttgtttcagg atgcaggaag ggtctgaagt tttttctaat ccatgcctgg aagaaaacaa
     5401 accaggcatt gtttttgctt ccctgaacca ttctgtcatt ggactgaact caagactggc
     5461 aagaaatgta aaagaagcac caacagaata tgcatccata tgtgtgagga gtggatcctg
     5521 gagccacccg cagttcgaga aaggtggagg ttccggaggt ggatcgggag gtggatcgtg
     5581 gagccacccg cagttcgaaa aataattcga gctccgtcga caagcttgcg gccgcactcg
     5641 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     5701 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     5761 tgag
//
