LOCUS       sequence-12180-e        8074 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 8074)
  AUTHORS   Maier CJ, Maier RH, Hintner H, Bauer JW, Onder K
  TITLE     Coupled yeast 2-hybrid-mammalian 2-hybrid reading-frame-independent 
            and site-specific recombinational cloning vector system.
  JOURNAL   Assay Drug Dev Technol. 2010 Oct;8(5):625-9. doi: 
            10.1089/adt.2009.0266. Epub 2010 Jun 1.
  PUBMED    20515414
REFERENCE   2  (bases 1 to 8074)
  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: 
            "10.1089/adt.2009.0266"; journalName: "Assay Drug Dev Technol"; 
            date: "2010-10"; volume: "8"; issue: "5"; pages: "625-9"
COMMENT     Sequence Label: pMBD-Gate3
FEATURES             Location/Qualifiers
     source          1..8074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     680..700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1005..1024
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1067..1085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1116..1556
                     /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    1600..1724
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1749..1754,1755..1772,1773..1779)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1833..2513
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(1899..1918)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2833..3138
                     /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     3017..3036
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3179..3303)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3389..3510
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3400..3419)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3454..3473
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3696..4151
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3783..3802)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3993..4014
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4223..4580
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4223..4414
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4246..4266)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4431..4566
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4922..5857
                     /codon_start=1
                     /product="luciferase from the anthozoan coelenterate 
                     Renilla reniformis (sea pansy)"
                     /label=Rluc
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     polyA_signal    5915..5963
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(6012..6031)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6131..6153
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6191..6209)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6277..6381
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6382..6450,6451..7242)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6600..6619)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7413..8001
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7902..7921
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
      661 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact
     1081 ataggctagc cagcttgaag caagcctcct gaaagatgaa gctactgtct tctatcgaac
     1141 aagcatgcga tatttgccga cttaaaaagc tcaagtgctc caaagaaaaa ccgaagtgcg
     1201 ccaagtgtct gaagaacaac tgggagtgtc gctactctcc caaaaccaaa aggtctccgc
     1261 tgactagggc acatctgaca gaagtggaat caaggctaga aagactggaa cagctatttc
     1321 tactgatttt tcctcgagaa gaccttgaca tgattttgaa aatggattct ttacaggata
     1381 taaaagcatt gttaacagga ttatttgtac aagataatgt gaataaagat gccgtcacag
     1441 atagattggc ttcagtggag actgatatgc ctctaacatt gagacagcat agaataagtg
     1501 cgacatcatc atcggaagag agtagtaaca aaggtcaaag acagttgact gtatcgccgg
     1561 aattcccggg gatccgtcga cttgacgcgt tgatatcaaa caagtttgta caaaaaagct
     1621 gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa
     1681 cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc gcattaggca
     1741 ccccaggctt tacactttat gcttccggct cgtataatgt gtggattttg agttaggatc
     1801 cgtcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca
     1861 ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc
     1921 aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga
     1981 aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc
     2041 atccggaatt ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc
     2101 cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc
     2161 acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa
     2221 acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct
     2281 gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg
     2341 ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc
     2401 aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac
     2461 agtactgcga tgagtggcag gcggggcgta atctagagga tccggcttac taaaagccag
     2521 ataacagtat gcgtatttgc gcgctgattt ttgcggtata agaatatata ctgatatgta
     2581 tacccgaagt atgtcaaaaa gaggtatgct atgaagcagc gtattacagt gacagttgac
     2641 agcgacagct atcagttgct caaggcatat atgatgtcaa tatctccggt ctggtaagca
     2701 caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg gaaaatcagg
     2761 aagggatggc tgaggtcgcc cggtttattg aaatgaacgg ctcttttgct gacgagaaca
     2821 ggggctggtg aaatgcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg
     2881 tttgtggatg tacagagtga tattattgac acgcccgggc gacggatggt gatccccctg
     2941 gccagtgcac gtctgctgtc agataaagtc ccccgtgaac tttacccggt ggtgcatatc
     3001 ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc
     3061 ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg
     3121 atgttctggg gaatataaat gtcaggctcc cttatacaca gccagtctgc aggtcgacca
     3181 tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat gcaaaatcta
     3241 atttaatata ttgatattta tatcatttta cgtttctcgt tcagctttct tgtacaaagt
     3301 ggttcgatat catctagagc ggccgcaggt acctgaataa ctaaggccgc ttccctttag
     3361 tgagggttaa tgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca
     3421 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     3481 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     3541 caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     3601 aaaatccgat aaggatcgat ccgggctggc gtaatagcga agaggcccgc accgatcgcc
     3661 cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag
     3721 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3781 cgctcctttc gctttcttcc cttcctttca cgccacgttc gccggctttc cccgtcaagc
     3841 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3901 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3961 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     4021 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     4081 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     4141 gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4201 atacgcggat cttccgtacc ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     4261 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4321 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4381 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4441 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4501 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4561 gaggcctagg cttttgcaaa aagcttgatt cttctgacac aacagtctcg aacttaaggc
     4621 tagaattctg gctacaggta agcgccccta aaatcccttt gggcacaatg tgtcctgagg
     4681 ggagaggcag cgacctgtag atgggacggg ggcactaacc ctcaggtttg gggcttctga
     4741 atgtgagtat cgccatgtaa gcccagtatt tggccaatct cagaaagctc ctggtccctg
     4801 gagggatgga gagagaaaaa caaacagctc ctggagcagg gagagtgctg gcctcttgct
     4861 ctccggctcc ctctgttgcc ctctggtttc tccccaggct cccggacgtc ctctagccac
     4921 catgacttcg aaagtttatg atccagaaca aaggaaacgg atgataactg gtccgcagtg
     4981 gtgggccaga tgtaaacaaa tgaatgttct tgattcattt attaattatt atgattcaga
     5041 aaaacatgca gaaaatgctg ttattttttt acatggtaac gcggcctctt cttatttatg
     5101 gcgacatgtt gtgccacata ttgagccagt agcgcggtgt attataccag accttattgg
     5161 tatgggcaaa tcaggcaaat ctggtaatgg ttcttatagg ttacttgatc attacaaata
     5221 tcttactgca tggtttgaac ttcttaattt accaaagaag atcatttttg tcggccatga
     5281 ttggggtgct tgtttggcat ttcattatag ctatgagcat caagataaga tcaaagcaat
     5341 agttcacgct gaaagtgtag tagatgtgat tgaatcatgg gatgaatggc ctgatattga
     5401 agaagatatt gcgttgatca aatctgaaga aggagaaaaa atggttttgg agaataactt
     5461 cttcgtggaa accatgttgc catcaaaaat catgagaaag ttagaaccag aagaatttgc
     5521 agcatatctt gaaccattca aagagaaagg tgaagttcgt cgtccaacat tatcatggcc
     5581 tcgtgaaatc ccgttagtaa aaggtggtaa acctgacgtt gtacaaattg ttaggaatta
     5641 taatgcttat ctacgtgcaa gtgatgattt accaaaaatg tttattgaat cggacccagg
     5701 attcttttcc aatgctattg ttgaaggtgc caagaagttt cctaatactg aatttgtcaa
     5761 agtaaaaggt cttcattttt cgcaagaaga tgcacctgat gaaatgggaa aatatatcaa
     5821 atcgttcgtt gagcgagttc tcaaaaatga acaataattc tagccctgaa taagtgataa
     5881 taagcggatg aatggcagaa attcgtcgaa gcgcaataaa atatctttat tttcattaca
     5941 tctgtgtgtt ggttttttgt gtgaatcgat agcgataagg atcggaagat ccgcgtatgg
     6001 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     6061 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     6121 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     6181 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     6241 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     6301 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     6361 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     6421 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     6481 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     6541 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     6601 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata
     6661 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     6721 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     6781 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     6841 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6901 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     6961 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     7021 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     7081 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     7141 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     7201 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     7261 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     7321 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     7381 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     7441 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     7501 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     7561 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     7621 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     7681 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     7741 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     7801 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     7861 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     7921 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7981 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     8041 tgctggcctt ttgctcacat ggctcgacag atct
//
