LOCUS       sequence-252116-        5742 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  generation of biotinylated human CD72 CTLD protein using bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5742)
  TITLE     Tsubata CD72, SmB, and U1-C plasmids
REFERENCE   2  (bases 1 to 5742)
  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: pET28aHisAvihCD72CTLD
FEATURES             Location/Qualifiers
     source          1..5742
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     55..74
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     158..177
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        158..176
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    177..201
                     /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             216..238
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             251..268
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             278..295
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             302..346
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             764..781
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(834..852)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      848..895
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      932..1387
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1019..1038)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1229..1250
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1479..2294)
                     /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(1479..2294)
                     /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     2202..2221
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2286..2305
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2416..3004
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2905..2924
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3158..3175
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3190..3329
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3255..3274)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3415..3437
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3431..3622)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4431..5513)
                     /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     5475..5494
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5514..5591)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
       61 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
      121 tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa
      181 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata
      241 taccatgtcc catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatag
      301 tggcctgaac gatatcttcg aggctcagaa aatcgaatgg cacgaacata tgtgtccgtc
      361 gggatggata atgcatcaga aaagctgctt ttacatctca cttacttcaa aaaattggca
      421 ggagagccaa aaacaatgtg aaactctgtc ttccaagctg gccacattca gtgaaattta
      481 tccacaatca cactcttact acttcttaaa ttcactgttg ccaaatggtg gttcagggaa
      541 ttcatattgg actggcctca gctctaacaa ggattggaag ttgactgatg atacacaacg
      601 cactaggact tatgctcaaa gctcaaaatg taacaaggta cataaaactt ggtcatggtg
      661 gacactggag tcagagtcat gtagaagttc tcttccctac atctgtgaga tgacagcttt
      721 caggtttcca gattgagtcg acaagcttgc ggccgcactc gagcaccacc accaccacca
      781 ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
      841 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
      901 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
      961 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1021 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1081 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1141 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     1201 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1261 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     1321 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     1381 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1441 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
     1501 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     1561 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     1621 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     1681 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     1741 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     1801 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     1861 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     1921 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     1981 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     2041 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     2101 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     2161 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     2221 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     2281 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     2341 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2401 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2461 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2521 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     2581 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2641 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2701 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2761 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2821 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2881 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2941 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3001 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3061 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3121 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3181 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     3241 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     3301 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     3361 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3421 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     3481 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     3541 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     3601 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     3661 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     3721 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     3781 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     3841 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     3901 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     3961 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     4021 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     4081 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     4141 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     4201 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     4261 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     4321 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     4381 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     4441 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4501 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     4561 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     4621 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     4681 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     4741 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     4801 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     4861 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     4921 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     4981 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     5041 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     5101 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     5161 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     5221 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     5281 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     5341 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     5401 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     5461 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     5521 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     5581 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     5641 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     5701 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cc
//
