LOCUS       sequence-12182-e        8073 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 8073)
  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 8073)
  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-Gate5
FEATURES             Location/Qualifiers
     source          1..8073
                     /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    1599..1723
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1764..2069)
                     /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
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1866..1885)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2411..3070)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2985..3004
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(3124..3130,3131..3148,3149..3154))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(3179..3303)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3388..3509
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3399..3418)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3453..3472
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3695..4150
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3782..3801)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3992..4013
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4222..4579
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4222..4413
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4245..4265)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4430..4565
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4921..5856
                     /codon_start=1
                     /product="luciferase from the anthozoan coelenterate 
                     Renilla reniformis (sea pansy)"
                     /label=Rluc
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     polyA_signal    5914..5962
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(6011..6030)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6130..6152
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6190..6208)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6276..6380
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6381..6449,6450..7241)
                     /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(6599..6618)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7412..8000
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7901..7920
                     /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 tgatatcaac cactttgtac aagaaagctg
     1621 aacgagaaac gtaaaatgat ataaatatca atatattaaa ttagattttg cataaaaaac
     1681 agactacata atactgtaaa acacaacata tccagtcact atggtcgacc tgcagactgg
     1741 ctgtgtataa gggagcctga catttatatt ccccagaaca tcaggttaat ggcgtttttg
     1801 atgtcatttt cgcggtggct gagatcagcc acttcttccc cgataacgga gaccggcaca
     1861 ctggccatat cggtggtcat catgcgccag ctttcatccc cgatatgcac caccgggtaa
     1921 agttcacggg agactttatc tgacagcaga cgtgcactgg ccagggggat caccatccgt
     1981 cgcccgggcg tgtcaataat atcactctgt acatccacaa acagacgata acggctctct
     2041 cttttatagg tgtaaacctt aaactgcatt tcaccagccc ctgttctcgt cagcaaaaga
     2101 gccgttcatt tcaataaacc gggcgacctc agccatccct tcctgatttt ccgctttcca
     2161 gcgttcggca cgcagacgac gggcttcatt ctgcatggtt gtgcttacca gaccggagat
     2221 attgacatca tatatgcctt gagcaactga tagctgtcgc tgtcaactgt cactgtaata
     2281 cgctgcttca tagcatacct ctttttgaca tacttcgggt atacatatca gtatatattc
     2341 ttataccgca aaaatcagcg cgcaaatacg catactgtta tctggctttt agtaagccgg
     2401 atccagatct ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca
     2461 ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca
     2521 gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt
     2581 ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga
     2641 aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca
     2701 catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg
     2761 atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata
     2821 tcaccagctc accgtctttc attgccatac ggaattccgg atgagcattc atcaggcggg
     2881 caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa
     2941 aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg
     3001 cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt
     3061 ttttctccat tttagcttcc ttagctcctg aaaatctcga cggatcctaa ctcaaaatcc
     3121 acacattata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gcggccgcca
     3181 tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat gcaaaatcta
     3241 atttaatata ttgatattta tatcatttta cgtttctcgt tcagcttttt tgtacaaact
     3301 tgttgatatc atctagagcg gccgcaggta cctgaataac taaggccgct tccctttagt
     3361 gagggttaat gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa
     3421 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     3481 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     3541 aggttcaggg ggagatgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
     3601 aaatccgata aggatcgatc cgggctggcg taatagcgaa gaggcccgca ccgatcgccc
     3661 ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc
     3721 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3781 gctcctttcg ctttcttccc ttcctttcac gccacgttcg ccggctttcc ccgtcaagct
     3841 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3901 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3961 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4021 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4081 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4141 cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4201 tacgcggatc ttccgtacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     4261 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4321 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4381 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4441 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4501 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     4561 aggcctaggc ttttgcaaaa agcttgattc ttctgacaca acagtctcga acttaaggct
     4621 agaattctgg ctacaggtaa gcgcccctaa aatccctttg ggcacaatgt gtcctgaggg
     4681 gagaggcagc gacctgtaga tgggacgggg gcactaaccc tcaggtttgg ggcttctgaa
     4741 tgtgagtatc gccatgtaag cccagtattt ggccaatctc agaaagctcc tggtccctgg
     4801 agggatggag agagaaaaac aaacagctcc tggagcaggg agagtgctgg cctcttgctc
     4861 tccggctccc tctgttgccc tctggtttct ccccaggctc ccggacgtcc tctagccacc
     4921 atgacttcga aagtttatga tccagaacaa aggaaacgga tgataactgg tccgcagtgg
     4981 tgggccagat gtaaacaaat gaatgttctt gattcattta ttaattatta tgattcagaa
     5041 aaacatgcag aaaatgctgt tattttttta catggtaacg cggcctcttc ttatttatgg
     5101 cgacatgttg tgccacatat tgagccagta gcgcggtgta ttataccaga ccttattggt
     5161 atgggcaaat caggcaaatc tggtaatggt tcttataggt tacttgatca ttacaaatat
     5221 cttactgcat ggtttgaact tcttaattta ccaaagaaga tcatttttgt cggccatgat
     5281 tggggtgctt gtttggcatt tcattatagc tatgagcatc aagataagat caaagcaata
     5341 gttcacgctg aaagtgtagt agatgtgatt gaatcatggg atgaatggcc tgatattgaa
     5401 gaagatattg cgttgatcaa atctgaagaa ggagaaaaaa tggttttgga gaataacttc
     5461 ttcgtggaaa ccatgttgcc atcaaaaatc atgagaaagt tagaaccaga agaatttgca
     5521 gcatatcttg aaccattcaa agagaaaggt gaagttcgtc gtccaacatt atcatggcct
     5581 cgtgaaatcc cgttagtaaa aggtggtaaa cctgacgttg tacaaattgt taggaattat
     5641 aatgcttatc tacgtgcaag tgatgattta ccaaaaatgt ttattgaatc ggacccagga
     5701 ttcttttcca atgctattgt tgaaggtgcc aagaagtttc ctaatactga atttgtcaaa
     5761 gtaaaaggtc ttcatttttc gcaagaagat gcacctgatg aaatgggaaa atatatcaaa
     5821 tcgttcgttg agcgagttct caaaaatgaa caataattct agccctgaat aagtgataat
     5881 aagcggatga atggcagaaa ttcgtcgaag cgcaataaaa tatctttatt ttcattacat
     5941 ctgtgtgttg gttttttgtg tgaatcgata gcgataagga tcggaagatc cgcgtatggt
     6001 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     6061 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     6121 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     6181 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     6241 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     6301 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     6361 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     6421 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     6481 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     6541 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6601 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     6661 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6721 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6781 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6841 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6901 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6961 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     7021 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     7081 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     7141 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     7201 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     7261 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     7321 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     7381 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     7441 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     7501 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     7561 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     7621 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     7681 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7741 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7801 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7861 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7921 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7981 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     8041 gctggccttt tgctcacatg gctcgacaga tct
//
