LOCUS       sequence-9922-ex        5219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5219)
  AUTHORS   Mon H, Sugahara R, Hong SM, Lee JM, Kamachi Y, Kawaguchi Y, Kusakabe
            T
  TITLE     Analysis of protein interactions with two-hybrid system in cultured 
            insect cells.
  JOURNAL   Anal Biochem. 2009 Sep 15;392(2):180-2. doi: 
            10.1016/j.ab.2009.05.033.
  PUBMED    19481053
REFERENCE   2  (bases 1 to 5219)
  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: 
            "10.1016/j.ab.2009.05"; journalName: "Anal Biochem"; date: 
            "2009-09-15- 15"; volume: "392"; issue: "2"; pages: "180-2"
COMMENT     Sequence Label: pIE2-DBD
FEATURES             Location/Qualifiers
     source          1..5219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..548
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 promoter
                     /note="strong constitutive baculovirus promoter for insect 
                     cell expression"
     regulatory      597..606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             636..725
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             753..1193
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     protein_bind    1194..1318
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1343..1348,1349..1366,1367..1373)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1427..2107
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(1493..1512)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2427..2732
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2611..2630
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2773..2897)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2927..2968
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2978..2995
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    3013..3142
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 poly(A) signal
                     /note="baculovirus polyadenylation signal"
     rep_origin      complement(3270..3858)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3350..3369)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4023..5213)
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     4842..4861
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 catgatgata aacaatgtat ggtgctaatg ttgcttcaac aacaattctg ttgaactgtg
       61 ttttcatgtt tgccaacaag cacctttata ctcggtggcc tccccaccac caactttttt
      121 gcactgcaaa aaaacacgct tttgcacgcg ggcccataca tagtacaaac tctacgtttc
      181 gtagactatt ttacataaat agtctacacc gttgtatacg ctccaaatac actaccacac
      241 attgaacctt tttgcagtgc aaaaaagtac gtgtcggcag tcacgtaggc cggccttatc
      301 gggtcgcgtc ctgtcacgta cgaatcacat tatcggaccg gacgagtgtt gtcttatcgt
      361 gacaggacgc cagcttcctg tgttgctaac cgcagccgga cgcaactcct tatcggaaca
      421 ggacgcgcct ccatatcagc cgcgcgttat ctcatgcgcg tgaccggaca cgaggcgccc
      481 gtcccgctta tcgcgcctat aaatacagcc cgcaacgatc tggtaaacac agttgaacag
      541 catctgttcg aatttaaagc ttggtaccga gatcgaggcc tgtctagaga agctccgcca
      601 ccatggatct ccaccgcggt ggaggccgca tcttttaccc atacgatgtt cctgactatg
      661 cgggctatcc ctatgacgtc ccggactatg caggatccta tccatatgac gttccagatt
      721 acgctgctca tggcggacac cggtatacta gtatgaagct actgtcttct atcgaacaag
      781 catgcgatat ttgccgactt aaaaagctca agtgctccaa agaaaaaccg aagtgcgcca
      841 agtgtctgaa gaacaactgg gagtgtcgct actctcccaa aaccaaaagg tctccgctga
      901 ctagggcaca tctgacagaa gtggaatcaa ggctagaaag actggaacag ctatttctac
      961 tgatttttcc tcgagaagac cttgacatga ttttgaaaat ggattcttta caggatataa
     1021 aagcattgtt aacaggatta tttgtacaag ataatgtgaa taaagatgcc gtcacagata
     1081 gattggcttc agtggagact gatatgcctc taacattgag acagcataga ataagtgcga
     1141 catcatcatc ggaagagagt agtaacaaag gtcaaagaca gttgactgta tcgacaagtt
     1201 tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga
     1261 ttttgcataa aaaacagact acataatact gtaaaacaca acatatccag tcactatggc
     1321 ggccgcatta ggcaccccag gctttacact ttatgcttcc ggctcgtata atgtgtggat
     1381 tttgagttag gatccgtcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat
     1441 cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt
     1501 tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt
     1561 aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg
     1621 cctgatgaat gctcatccgg aattccgtat ggcaatgaaa gacggtgagc tggtgatatg
     1681 ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct
     1741 ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc
     1801 gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt
     1861 ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa
     1921 cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat
     1981 gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct
     2041 taatgaatta caacagtact gcgatgagtg gcaggcgggg cgtaatctag aggatccggc
     2101 ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg atttttgcgg tataagaata
     2161 tatactgata tgtatacccg aagtatgtca aaaagaggta tgctatgaag cagcgtatta
     2221 cagtgacagt tgacagcgac agctatcagt tgctcaaggc atatatgatg tcaatatctc
     2281 cggtctggta agcacaacca tgcagaatga agcccgtcgt ctgcgtgccg aacgctggaa
     2341 agcggaaaat caggaaggga tggctgaggt cgcccggttt attgaaatga acggctcttt
     2401 tgctgacgag aacaggggct ggtgaaatgc agtttaaggt ttacacctat aaaagagaga
     2461 gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccc gggcgacgga
     2521 tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtcccccgt gaactttacc
     2581 cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc
     2641 cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa
     2701 acgccattaa cctgatgttc tggggaatat aaatgtcagg ctcccttata cacagccagt
     2761 ctgcaggtcg accatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt
     2821 ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcagct
     2881 ttcttgtaca aagtggtgac gtaagctaga gggcccgcgg ttcgaaggta agcctatccc
     2941 taaccctctc ctcggtctcg attctacgcg taccggtcat catcaccatc accattgagt
     3001 ttatctgact aaatcttagt ttgtattgtc atgttttaat acaatatgtt atgtttaaat
     3061 atgtttttaa taaattttat aaaataattt caacttttat tgtaacaaca ttgtccattt
     3121 acacactcct ttcaagcgcg tgggatcgat gctcactcaa aggcggtaat acggttatcc
     3181 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3241 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3301 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3361 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3421 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     3481 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3541 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3601 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3661 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3721 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3781 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3841 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3901 aacgaaaact cacgttaagg gattttggtc atgcgaaaca cgcacggcgc gcgcacgcag
     3961 cttagcacaa acgcgtcgtt gcagagtttc gaatccgtta gcgaggtgcc gccggcttcc
     4021 attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg
     4081 tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa
     4141 atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat
     4201 ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg
     4261 ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc
     4321 gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc
     4381 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     4441 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     4501 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     4561 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     4621 ttgaaggctc tcaagggcat cggtcgacgc tctcccttat gcgactcctg cattaggaag
     4681 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     4741 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     4801 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     4861 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     4921 atccacagga cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa
     4981 gcgagcagga ctgggcggcg gccaaagcgg tcggacagtg ctccgagaac gggtgcgcat
     5041 agaaattgca tcaacgcata tagcgctagc agcacgccat agtgactggc gatgctgtcg
     5101 gaatggacga tatcccgcaa gaggcccggc agtaccggca taaccaagcc tatgcctaca
     5161 gcatccaggg tgacggtgcc gaggatgacg atgagcgcat tgttagattt catagatct
//
