LOCUS       sequence-252110-        5743 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 5743)
  TITLE     Tsubata CD72, SmB, and U1-C plasmids
REFERENCE   2  (bases 1 to 5743)
  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"
COMMENT     Sequence Label: pET28aHisAviCD72aCTLD
FEATURES             Location/Qualifiers
     source          1..5743
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     555..574
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(594..671)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     877..896
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     980..999
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        980..998
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    999..1023
                     /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             1038..1060
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1073..1090
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1100..1117
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1124..1168
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1587..1604
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1657..1675)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1671..1718
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1755..2210
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1842..1861)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2052..2073
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2302..3117)
                     /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(2302..3117)
                     /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     3025..3044
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3109..3128
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3239..3827
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3728..3747
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3981..3998
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4013..4152
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4078..4097)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4238..4260
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4254..4445)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5254..5743,1..593))
                     /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 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
       61 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
      121 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
      181 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
      241 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
      301 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
      361 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
      421 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
      481 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
      541 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
      601 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
      661 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
      721 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
      781 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
      841 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
      901 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
      961 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     1021 tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgt cccatcatca
     1081 tcatcatcac agcagcggcc tggtgccgcg cggcagccat agtggcctga acgatatctt
     1141 cgaggctcag aaaatcgaat ggcacgaaca tatgtgtcca tgcggatgga ttccatatca
     1201 ggaaaggtgc ttttacatct cacataccct cagaagtctg gaggagagcc aaaaatactg
     1261 cacatctctg tcctccaaac tggcagcatt cgatgaacct tctaagtatt actatgaagt
     1321 ttctctgccc agcggcttag aggagttgct agatcgttcg aagtcatatt ggatacagat
     1381 gagcaagaag tggaggcatg actatgactc tcaaagccga tattgtgaca agataaaaaa
     1441 atattaccag aagtggaaaa gaacattttc tgagtgtgca gagcttcacc cctgcatttg
     1501 tgagtcggag gctttcaggt ttcctgatgg gatccatctg aactgagaat tcgagctccg
     1561 tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta
     1621 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     1681 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     1741 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1801 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1861 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     1921 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     1981 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2041 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2101 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2161 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     2221 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2281 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     2341 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     2401 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     2461 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     2521 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     2581 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     2641 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     2701 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     2761 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     2821 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     2881 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     2941 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     3001 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     3061 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     3121 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     3181 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3241 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3301 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3361 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3421 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3481 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3541 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3601 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3661 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3721 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3781 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3841 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3901 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3961 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     4021 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     4081 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     4141 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4201 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4261 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     4321 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     4381 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     4441 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     4501 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     4561 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     4621 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     4681 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     4741 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     4801 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     4861 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     4921 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     4981 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     5041 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     5101 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     5161 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     5221 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5281 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5341 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     5401 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     5461 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     5521 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     5581 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     5641 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     5701 cggctgaatt tgattgcgag tgagatattt atgccagcca gcc
//
