LOCUS       sequence-252108-        5734 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  generation of biotinylated mouse CD72c CTLD protein using bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5734)
  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 5734)
  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: pET28aAviHisCD72cCTLD
FEATURES             Location/Qualifiers
     source          1..5734
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     90..109
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     193..212
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        193..211
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    212..236
                     /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             251..273
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             286..330
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             346..363
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             373..390
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             791..808
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(861..879)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      875..922
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      959..1414
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1046..1065)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1256..1277
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1506..2321)
                     /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(1506..2321)
                     /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     2229..2248
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2313..2332
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2443..3031
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2932..2951
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3185..3202
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3217..3356
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3282..3301)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3442..3464
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3458..3649)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4458..5540)
                     /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     5502..5521
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5541..5618)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
       61 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
      121 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
      181 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
      241 tagaaataat tttgtttaac tttaagaagg agatatacca tgtccggcct gaacgatatc
      301 ttcgaggctc agaaaatcga atggcacgaa gccatgggca gcagccatca tcatcatcat
      361 cacagcagcg gcctggtgcc gcgcggcagc catatgtgtc catgcggatg gattccatat
      421 cagaaaaggt gcttttacat ctcacatacc ctcagaagtc tggaggaaag ccaaaaatac
      481 tgcacatctc tgtcctccaa attggcagca ttcaatgacc ctcctaagta tttctatcaa
      541 gtttctctgc ccagcggctt agaggagttg ctagatcgtt tgaagtcata ttggatacag
      601 gagatgagca agaagtggag ctcatattgt gacatgatac aaatatataa caagaagtgg
      661 gcaagaacat tttccgagtg tgcagagctt cacccctgca tttgtgagtt ggaggctttc
      721 aggtttcctg atgggatcca tctgaactga gaattcgagc tccgtcgaca agcttgcggc
      781 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
      841 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
      901 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
      961 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1021 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1081 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1141 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     1201 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1261 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     1321 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     1381 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     1441 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     1501 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     1561 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     1621 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     1681 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     1741 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     1801 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     1861 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     1921 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     1981 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     2041 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2101 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2161 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     2221 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     2281 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     2341 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     2401 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2461 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2521 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2581 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2641 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2701 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2761 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2821 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     2881 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2941 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3001 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3061 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3121 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3181 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3241 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     3301 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     3361 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3421 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3481 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     3541 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     3601 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     3661 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     3721 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     3781 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     3841 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     3901 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     3961 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     4021 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     4081 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     4141 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     4201 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     4261 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     4321 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     4381 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     4441 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4501 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     4561 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     4621 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     4681 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     4741 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     4801 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     4861 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     4921 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     4981 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     5041 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     5101 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     5161 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     5221 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     5281 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     5341 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     5401 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     5461 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     5521 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     5581 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     5641 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     5701 cgcaaggaat ggtgcatgca aggagatggc gccc
//
