LOCUS       sequence-252106-        5752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  generation of biotinylated mouse CD72a CTLD protein using bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5752)
  AUTHORS   Akatsu C, Shinagawa K, Numoto N, Liu Z, Ucar AK, Aslam M, Phoon S, 
            Adachi T, Furukawa K, Ito N, Tsubata T
  TITLE     CD72 negatively regulates B lymphocyte responses to the 
            lupus-related endogenous toll-like receptor 7 ligand Sm/RNP.
  JOURNAL   J Exp Med. 2016 Nov 14;213(12):2691-2706. doi: 10.1084/jem.20160560.
            Epub 2016 Oct 24.
  PUBMED    27810925
REFERENCE   2  (bases 1 to 5752)
  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"; doi: 
            "10.1084/jem.20160560"; journalName: "J Exp Med"; date: "2016-11-14-
            14"; volume: "213"; issue: "12"; pages: "2691-2706"
COMMENT     Sequence Label: pET28aAviHisCD72aCTLD
FEATURES             Location/Qualifiers
     source          1..5752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     925..944
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(964..1041)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1247..1266
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1350..1369
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1350..1368
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1369..1393
                     /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             1408..1430
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1443..1487
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1503..1520
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1530..1547
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1966..1983
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2036..2054)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2050..2097
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2134..2589
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2221..2240)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2431..2452
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2681..3496)
                     /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(2681..3496)
                     /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     3404..3423
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3488..3507
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3618..4206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4107..4126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4360..4377
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4392..4531
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4457..4476)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4617..4639
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4633..4824)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5633..5752,1..963))
                     /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"
ORIGIN
        1 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
       61 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
      121 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
      181 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
      241 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
      301 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
      361 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
      421 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
      481 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
      541 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
      601 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
      661 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
      721 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
      781 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
      841 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
      901 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
      961 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     1021 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     1081 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     1141 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     1201 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     1261 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     1321 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     1381 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgt
     1441 ccggcctgaa cgatatcttc gaggctcaga aaatcgaatg gcacgaagcc atgggcagca
     1501 gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat atgtgtccat
     1561 gcggatggat tccatatcag gaaaggtgct tttacatctc acataccctc agaagtctgg
     1621 aggagagcca aaaatactgc acatctctgt cctccaaact ggcagcattc gatgaacctt
     1681 ctaagtatta ctatgaagtt tctctgccca gcggcttaga ggagttgcta gatcgttcga
     1741 agtcatattg gatacagatg agcaagaagt ggaggcatga ctatgactct caaagccgat
     1801 attgtgacaa gataaaaaaa tattaccaga agtggaaaag aacattttct gagtgtgcag
     1861 agcttcaccc ctgcatttgt gagtcggagg ctttcaggtt tcctgatggg atccatctga
     1921 actgagaatt cgagctccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac
     1981 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     2041 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     2101 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     2161 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2221 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2281 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2341 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2401 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2461 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     2521 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     2581 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2641 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     2701 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     2761 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     2821 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     2881 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     2941 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     3001 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     3061 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     3121 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     3181 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     3241 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     3301 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     3361 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     3421 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     3481 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     3541 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3601 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3661 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3721 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3781 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3841 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3901 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3961 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4021 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4081 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4141 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4201 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4261 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4321 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4381 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4441 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     4501 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     4561 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     4621 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     4681 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     4741 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     4801 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     4861 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     4921 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     4981 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     5041 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     5101 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     5161 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     5221 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     5281 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     5341 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     5401 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     5461 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     5521 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     5581 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     5641 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5701 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt ga
//
