LOCUS       sequence-9923-ex        5316 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 5316)
  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 5316)
  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-AD
FEATURES             Location/Qualifiers
     source          1..5316
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        10..557
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 promoter
                     /note="strong constitutive baculovirus promoter for insect 
                     cell expression"
     CDS             622..642
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      652..661
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             661..726
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             748..1290
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     protein_bind    1300..1424
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1449..1454,1455..1472,1473..1479)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1533..2213
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(1599..1618)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2533..2838
                     /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     2717..2736
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2879..3003)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3033..3074
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3084..3101
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    3119..3248
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 poly(A) signal
                     /note="baculovirus polyadenylation signal"
     rep_origin      complement(3376..3964)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3456..3475)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4129..5316,1..3))
                     /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     4948..4967
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 catagatctc atgatgataa acaatgtatg gtgctaatgt tgcttcaaca acaattctgt
       61 tgaactgtgt tttcatgttt gccaacaagc acctttatac tcggtggcct ccccaccacc
      121 aacttttttg cactgcaaaa aaacacgctt ttgcacgcgg gcccatacat agtacaaact
      181 ctacgtttcg tagactattt tacataaata gtctacaccg ttgtatacgc tccaaataca
      241 ctaccacaca ttgaaccttt ttgcagtgca aaaaagtacg tgtcggcagt cacgtaggcc
      301 ggccttatcg ggtcgcgtcc tgtcacgtac gaatcacatt atcggaccgg acgagtgttg
      361 tcttatcgtg acaggacgcc agcttcctgt gttgctaacc gcagccggac gcaactcctt
      421 atcggaacag gacgcgcctc catatcagcc gcgcgttatc tcatgcgcgt gaccggacac
      481 gaggcgcccg tcccgcttat cgcgcctata aatacagccc gcaacgatct ggtaaacaca
      541 gttgaacagc atctgttcga atttaaagct tggtaccgag atcgaggcct gtctagagaa
      601 gcttgttcga atctcgagat gcctaaaaag aagcgtaaag tcgagtactc cgccaccatg
      661 gactacaaag accatgacgg tgattataaa gatcatgaca tcgattacaa ggatgacgat
      721 gacaagcacc ggtatactag tgaattccct tctgggcaaa tctcaaacca ggccctggcc
      781 ttagcaccgt cctctgcccc agtccttgcc cagaccatgg tcccttcctc agccatggta
      841 cctctggctc agcccccagc tcctgcccca gttctaaccc cgggtcctcc ccagtccctg
      901 tctgcacctg ttccaaagag cacccaggct ggggaaggca cgctgtcgga agccctgctg
      961 cacctgcagt ttgatgctga tgaagacttg ggggccttgc ttggcaacag cacagaccca
     1021 ggagtgttca cagacctggc atctgtggac aactcagagt ttcagcagct cctgaaccag
     1081 ggtgtgtcca tgtctcactc cacagctgag cccatgctga tggagtaccc tgaagctata
     1141 actcgcctgg tgacagggtc ccagaggccc cctgacccag ctcccacacc cctggggacc
     1201 tcggggcttc ccaatggtct ctccggagat gaagacttct cctccattgc ggacatggac
     1261 ttctctgctc tgctgagtca gatcagctcc agcggccaaa caagtttgta caaaaaagct
     1321 gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa
     1381 cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc gcattaggca
     1441 ccccaggctt tacactttat gcttccggct cgtataatgt gtggattttg agttaggatc
     1501 cgtcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca
     1561 ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc
     1621 aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga
     1681 aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc
     1741 atccggaatt ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc
     1801 cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc
     1861 acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa
     1921 acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct
     1981 gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg
     2041 ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc
     2101 aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac
     2161 agtactgcga tgagtggcag gcggggcgta atctagagga tccggcttac taaaagccag
     2221 ataacagtat gcgtatttgc gcgctgattt ttgcggtata agaatatata ctgatatgta
     2281 tacccgaagt atgtcaaaaa gaggtatgct atgaagcagc gtattacagt gacagttgac
     2341 agcgacagct atcagttgct caaggcatat atgatgtcaa tatctccggt ctggtaagca
     2401 caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg gaaaatcagg
     2461 aagggatggc tgaggtcgcc cggtttattg aaatgaacgg ctcttttgct gacgagaaca
     2521 ggggctggtg aaatgcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg
     2581 tttgtggatg tacagagtga tattattgac acgcccgggc gacggatggt gatccccctg
     2641 gccagtgcac gtctgctgtc agataaagtc ccccgtgaac tttacccggt ggtgcatatc
     2701 ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc
     2761 ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg
     2821 atgttctggg gaatataaat gtcaggctcc cttatacaca gccagtctgc aggtcgacca
     2881 tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat gcaaaatcta
     2941 atttaatata ttgatattta tatcatttta cgtttctcgt tcagctttct tgtacaaagt
     3001 ggtgacgtaa gctagagggc ccgcggttcg aaggtaagcc tatccctaac cctctcctcg
     3061 gtctcgattc tacgcgtacc ggtcatcatc accatcacca ttgagtttat ctgactaaat
     3121 cttagtttgt attgtcatgt tttaatacaa tatgttatgt ttaaatatgt ttttaataaa
     3181 ttttataaaa taatttcaac ttttattgta acaacattgt ccatttacac actcctttca
     3241 agcgcgtggg atcgatgctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3301 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3361 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3421 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3481 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3541 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3601 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3661 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3721 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3781 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3841 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3901 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3961 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4021 ttaagggatt ttggtcatgc gaaacacgca cggcgcgcgc acgcagctta gcacaaacgc
     4081 gtcgttgcag agtttcgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt
     4141 ggcccggctc catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc
     4201 tacaatccat gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat
     4261 cagcggtcca atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc
     4321 ctgatggtcg tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc
     4381 gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag
     4441 caagacgtag cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa
     4501 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     4561 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     4621 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     4681 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     4741 gggcatcggt cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     4801 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     4861 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     4921 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     4981 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcc acaggacggg
     5041 tgtggtcgcc atgatcgcgt agtcgatagt ggctccaagt agcgaagcga gcaggactgg
     5101 gcggcggcca aagcggtcgg acagtgctcc gagaacgggt gcgcatagaa attgcatcaa
     5161 cgcatatagc gctagcagca cgccatagtg actggcgatg ctgtcggaat ggacgatatc
     5221 ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac
     5281 ggtgccgagg atgacgatga gcgcattgtt agattt
//
