LOCUS       sequence-12166-e        7280 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 7280)
  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 7280)
  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: pMAD-Gate6
FEATURES             Location/Qualifiers
     source          1..7280
                     /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             1149..1169
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    1363..1487
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1528..1833)
                     /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     complement(1630..1649)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2153..2833)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     2748..2767
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2887..2893,2894..2911,2912..2917))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2942..3066)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3152..3273
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3163..3182)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3217..3236
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3407..3862
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3494..3513)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3704..3725
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3877..4234
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3877..4068
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3900..3920)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4085..4220
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4285..5079
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4339..4358)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4949..4968
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5143..5191
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5218..5237)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5337..5359
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5397..5415)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5483..5587
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5588..5656,5657..6448)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5806..5825)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6619..7207
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7108..7127
                     /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 aagcatgccc aaaaaagaag agaaaggtag atgaattccc ggggatctcg acggcccccc
     1201 cgaccgatgt cagcctgggg gacgagctcc acttagacgg cgaggacgtg gcgatggcgc
     1261 atgccgacgc gctagacgat ttcgatctgg acatgttggg ggacggggat tccccgggtc
     1321 cgggatcgcc agggatccgt cgacttgacg cgttgatatc gaaccacttt gtacaagaaa
     1381 gctgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa
     1441 aaacagacta cataatactg taaaacacaa catatccagt cactatggtc gacctgcaga
     1501 ctggctgtgt ataagggagc ctgacattta tattccccag aacatcaggt taatggcgtt
     1561 tttgatgtca ttttcgcggt ggctgagatc agccacttct tccccgataa cggagaccgg
     1621 cacactggcc atatcggtgg tcatcatgcg ccagctttca tccccgatat gcaccaccgg
     1681 gtaaagttca cgggggactt tatctgacag cagacgtgca ctggccaggg ggatcaccat
     1741 ccgtcgcccg ggcgtgtcaa taatatcact ctgtacatcc acaaacagac gataacggct
     1801 ctctctttta taggtgtaaa ccttaaactg catttcacca gcccctgttc tcgtcagcaa
     1861 aagagccgtt catttcaata aaccgggcga cctcagccat cccttcctga ttttccgctt
     1921 tccagcgttc ggcacgcaga cgacgggctt cattctgcat ggttgtgctt accagaccgg
     1981 agatattgac atcatatatg ccttgagcaa ctgatagctg tcgctgtcaa ctgtcactgt
     2041 aatacgctgc ttcatagcat acctcttttt gacatacttc gggtatacat atcagtatat
     2101 attcttatac cgcaaaaatc agcgcgcaaa tacgcatact gttatctggc ttttagtaag
     2161 ccggatcctc tagattacgc cccgcctgcc actcatcgca gtactgttgt aattcattaa
     2221 gcattctgcc gacatggaag ccatcacaaa cggcatgatg aacctgaatc gccagcggca
     2281 tcagcacctt gtcgccttgc gtataatatt tgcccatggt gaaaacgggg gcgaagaagt
     2341 tgtccatatt ggccacgttt aaatcaaaac tggtgaaact cacccaggga ttggctgaga
     2401 cgaaaaacat attctcaata aaccctttag ggaaataggc caggttttca ccgtaacacg
     2461 ccacatcttg cgaatatatg tgtagaaact gccggaaatc gtcgtggtat tcactccaga
     2521 gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta acaagggtga acactatccc
     2581 atatcaccag ctcaccgtct ttcattgcca tacggaattc cggatgagca ttcatcaggc
     2641 gggcaagaat gtgaataaag gccggataaa acttgtgctt atttttcttt acggtcttta
     2701 aaaaggccgt aatatccagc tgaacggtct ggttataggt acattgagca actgactgaa
     2761 atgcctcaaa atgttcttta cgatgccatt gggatatatc aacggtggta tatccagtga
     2821 tttttttctc cattttagct tccttagctc ctgaaaatct cgacggatcc taactcaaaa
     2881 tccacacatt atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgcggccg
     2941 ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat
     3001 ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt ttttgtacaa
     3061 acttgtttga tatcatctag agcggccgca ggtacctgaa taactaaggc cgcttccctt
     3121 tagtgagggt taatgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc
     3181 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     3241 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     3301 tttcaggttc agggggagat gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     3361 ggtaaaatcc gataaggatc gattccggag cctgaatggc gaatggacgc gccctgtagc
     3421 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3481 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3541 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3601 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3661 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3721 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3781 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     3841 aaaatattaa cgcttacaat ttcgcctgtg taccttctga ggcggaaaga accagctgtg
     3901 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3961 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     4021 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     4081 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4141 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     4201 aggaggcttt tttggaggcc taggcttttg caaaaagctt gattcttctg acacaacagt
     4261 ctcgaactta aggctagagc caccatgatt gaacaagatg gattgcacgc aggttctccg
     4321 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     4381 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4441 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     4501 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4561 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4621 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4681 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4741 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4801 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4861 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4921 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4981 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     5041 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     5101 tgaccgacca agcgacgccc aacctgccat cacgatggcc gcaataaaat atctttattt
     5161 tcattacatc tgtgtgttgg ttttttgtgt gaagatccgc gtatggtgca ctctcagtac
     5221 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     5281 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5341 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     5401 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     5461 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5521 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5581 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5641 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5701 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5761 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5821 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5881 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5941 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     6001 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     6061 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     6121 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     6181 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     6241 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     6301 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     6361 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6421 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6481 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6541 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6601 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6661 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6721 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6781 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6841 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6901 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6961 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     7021 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     7081 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     7141 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     7201 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     7261 tcacatggct cgacagatct
//
