LOCUS       sequence-12181-e        8071 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 8071)
  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 8071)
  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-Gate4
FEATURES             Location/Qualifiers
     source          1..8071
                     /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    1598..1722
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1763..2068)
                     /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(1865..1884)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2410..3069)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2984..3003
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(3123..3129,3130..3147,3148..3153))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(3178..3302)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3386..3507
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3397..3416)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3451..3470
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3693..4148
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3780..3799)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3990..4011
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4220..4577
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4220..4411
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4243..4263)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4428..4563
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4919..5854
                     /codon_start=1
                     /product="luciferase from the anthozoan coelenterate 
                     Renilla reniformis (sea pansy)"
                     /label=Rluc
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     polyA_signal    5912..5960
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(6009..6028)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6128..6150
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6188..6206)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6274..6378
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6379..6447,6448..7239)
                     /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(6597..6616)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7410..7998
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7899..7918
                     /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 tgatatcacc actttgtaca agaaagctga
     1621 acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca
     1681 gactacataa tactgtaaaa cacaacatat ccagtcacta tggtcgacct gcagactggc
     1741 tgtgtataag ggagcctgac atttatattc cccagaacat caggttaatg gcgtttttga
     1801 tgtcattttc gcggtggctg agatcagcca cttcttcccc gataacggag accggcacac
     1861 tggccatatc ggtggtcatc atgcgccagc tttcatcccc gatatgcacc accgggtaaa
     1921 gttcacggga gactttatct gacagcagac gtgcactggc cagggggatc accatccgtc
     1981 gcccgggcgt gtcaataata tcactctgta catccacaaa cagacgataa cggctctctc
     2041 ttttataggt gtaaacctta aactgcattt caccagcccc tgttctcgtc agcaaaagag
     2101 ccgttcattt caataaaccg ggcgacctca gccatccctt cctgattttc cgctttccag
     2161 cgttcggcac gcagacgacg ggcttcattc tgcatggttg tgcttaccag accggagata
     2221 ttgacatcat atatgccttg agcaactgat agctgtcgct gtcaactgtc actgtaatac
     2281 gctgcttcat agcatacctc tttttgacat acttcgggta tacatatcag tatatattct
     2341 tataccgcaa aaatcagcgc gcaaatacgc atactgttat ctggctttta gtaagccgga
     2401 tccacgcgtt tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat
     2461 tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag
     2521 caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga agaagttgtc
     2581 catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg ctgagacgaa
     2641 aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac
     2701 atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga
     2761 tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat
     2821 caccagctca ccgtctttca ttgccatacg gaattccgga tgagcattca tcaggcgggc
     2881 aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa
     2941 ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc
     3001 ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt
     3061 tttctccatt ttagcttcct tagctcctga aaatctcgac ggatcctaac tcaaaatcca
     3121 cacattatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg cggccgccat
     3181 agtgactgga tatgttgtgt tttacagtat tatgtagtct gttttttatg caaaatctaa
     3241 tttaatatat tgatatttat atcattttac gtttctcgtt cagctttttt gtacaaactt
     3301 gtgatatcat ctagagcggc cgcaggtacc tgaataacta aggccgcttc cctttagtga
     3361 gggttaatgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact
     3421 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     3481 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     3541 gttcaggggg agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa
     3601 atccgataag gatcgatccg ggctggcgta atagcgaaga ggcccgcacc gatcgccctt
     3661 cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc
     3721 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3781 tcctttcgct ttcttccctt cctttcacgc cacgttcgcc ggctttcccc gtcaagctct
     3841 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3901 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3961 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4021 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4081 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     4141 tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4201 cgcggatctt ccgtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     4261 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     4321 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     4381 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     4441 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     4501 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     4561 gcctaggctt ttgcaaaaag cttgattctt ctgacacaac agtctcgaac ttaaggctag
     4621 aattctggct acaggtaagc gcccctaaaa tccctttggg cacaatgtgt cctgagggga
     4681 gaggcagcga cctgtagatg ggacgggggc actaaccctc aggtttgggg cttctgaatg
     4741 tgagtatcgc catgtaagcc cagtatttgg ccaatctcag aaagctcctg gtccctggag
     4801 ggatggagag agaaaaacaa acagctcctg gagcagggag agtgctggcc tcttgctctc
     4861 cggctccctc tgttgccctc tggtttctcc ccaggctccc ggacgtcctc tagccaccat
     4921 gacttcgaaa gtttatgatc cagaacaaag gaaacggatg ataactggtc cgcagtggtg
     4981 ggccagatgt aaacaaatga atgttcttga ttcatttatt aattattatg attcagaaaa
     5041 acatgcagaa aatgctgtta tttttttaca tggtaacgcg gcctcttctt atttatggcg
     5101 acatgttgtg ccacatattg agccagtagc gcggtgtatt ataccagacc ttattggtat
     5161 gggcaaatca ggcaaatctg gtaatggttc ttataggtta cttgatcatt acaaatatct
     5221 tactgcatgg tttgaacttc ttaatttacc aaagaagatc atttttgtcg gccatgattg
     5281 gggtgcttgt ttggcatttc attatagcta tgagcatcaa gataagatca aagcaatagt
     5341 tcacgctgaa agtgtagtag atgtgattga atcatgggat gaatggcctg atattgaaga
     5401 agatattgcg ttgatcaaat ctgaagaagg agaaaaaatg gttttggaga ataacttctt
     5461 cgtggaaacc atgttgccat caaaaatcat gagaaagtta gaaccagaag aatttgcagc
     5521 atatcttgaa ccattcaaag agaaaggtga agttcgtcgt ccaacattat catggcctcg
     5581 tgaaatcccg ttagtaaaag gtggtaaacc tgacgttgta caaattgtta ggaattataa
     5641 tgcttatcta cgtgcaagtg atgatttacc aaaaatgttt attgaatcgg acccaggatt
     5701 cttttccaat gctattgttg aaggtgccaa gaagtttcct aatactgaat ttgtcaaagt
     5761 aaaaggtctt catttttcgc aagaagatgc acctgatgaa atgggaaaat atatcaaatc
     5821 gttcgttgag cgagttctca aaaatgaaca ataattctag ccctgaataa gtgataataa
     5881 gcggatgaat ggcagaaatt cgtcgaagcg caataaaata tctttatttt cattacatct
     5941 gtgtgttggt tttttgtgtg aatcgatagc gataaggatc ggaagatccg cgtatggtgc
     6001 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     6061 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     6121 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     6181 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     6241 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     6301 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6361 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6421 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6481 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6541 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6601 tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac
     6661 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6721 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6781 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6841 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6901 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6961 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     7021 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     7081 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     7141 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     7201 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7261 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7321 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7381 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7441 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     7501 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     7561 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7621 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7681 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7741 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7801 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7861 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7921 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7981 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     8041 tggccttttg ctcacatggc tcgacagatc t
//
