LOCUS       sequence-356708-        5764 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of 10*His tagged human BTLA intracellular domain 
            (AA190-289)-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 Apr 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)-TwinStrep
FEATURES             Location/Qualifiers
     source          1..5764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      66..521
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(153..172)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     363..384
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(613..1428)
                     /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(613..1428)
                     /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     1336..1355
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1420..1439
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1550..2138
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2039..2058
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2292..2309
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2324..2463
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2389..2408)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2549..2571
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2565..2756)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3565..4647)
                     /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     4609..4628
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4648..4725)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4931..4950
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5034..5053
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5034..5052
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5053..5077
                     /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             5092..5114
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5133..5162
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5172..5189
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5193..5225
                     /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             5538..5561
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5598..5621
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5662..5679
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5732..5750)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(5746..5764,1..29)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
       61 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
      121 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
      181 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
      241 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
      301 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
      361 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
      421 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
      481 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
      541 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
      601 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
      661 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
      721 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
      781 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
      841 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
      901 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
      961 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     1021 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     1081 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     1141 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     1201 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     1261 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     1321 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     1381 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     1441 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     1501 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1561 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1621 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1681 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     1741 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     1801 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     1861 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     1921 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     1981 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2041 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2101 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2161 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2221 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2281 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     2341 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     2401 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     2461 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     2521 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     2581 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     2641 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     2701 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     2761 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     2821 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     2881 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     2941 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     3001 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     3061 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     3121 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     3181 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     3241 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     3301 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     3361 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     3421 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     3481 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     3541 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3601 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     3661 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     3721 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     3781 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     3841 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     3901 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     3961 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     4021 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     4081 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     4141 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     4201 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     4261 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     4321 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     4381 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     4441 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     4501 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     4561 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     4621 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     4681 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     4741 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     4801 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     4861 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     4921 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     4981 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     5041 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     5101 ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcat catcaccatc
     5161 acagcagcgg cctggtgccg cgcggcagcc atatggctag catgactggt ggacagcaaa
     5221 tgggtcgcgg agacacagca ggaagggaaa ttaacctggt tgatgctcac cttaagagtg
     5281 agcaaacaga agcaagcacc aggcaaaatt cccaagtact gctatcagaa actggaattt
     5341 atgataatga ccctgacctt tgtttcagga tgcaggaagg gtctgaagtt tattctaatc
     5401 catgcctgga agaaaacaaa ccaggcattg tttatgcttc cctgaaccat tctgtcattg
     5461 gactgaactc aagactggca agaaatgtaa aagaagcacc aacagaatat gcatccatat
     5521 gtgtgaggag tggatcctgg agccacccgc agttcgagaa aggtggaggt tccggaggtg
     5581 gatcgggagg tggatcgtgg agccacccgc agttcgaaaa ataattcgag ctccgtcgac
     5641 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     5701 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     5761 gggg
//
