LOCUS       sequence-12164-e        7277 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 7277)
  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 7277)
  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-Gate4
FEATURES             Location/Qualifiers
     source          1..7277
                     /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    1361..1485
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1526..1831)
                     /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(1628..1647)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2173..2832)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2747..2766
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2886..2892,2893..2910,2911..2916))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2941..3065)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3149..3270
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3160..3179)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3214..3233
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3404..3859
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3491..3510)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3701..3722
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3874..4231
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3874..4065
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3897..3917)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4082..4217
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4282..5076
                     /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(4336..4355)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4946..4965
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5140..5188
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5215..5234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5334..5356
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5394..5412)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5480..5584
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5585..5653,5654..6445)
                     /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(5803..5822)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6616..7204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7105..7124
                     /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 accactttgt acaagaaagc
     1381 tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt tgcataaaaa
     1441 acagactaca taatactgta aaacacaaca tatccagtca ctatggtcga cctgcagact
     1501 ggctgtgtat aagggagcct gacatttata ttccccagaa catcaggtta atggcgtttt
     1561 tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc cccgataacg gagaccggca
     1621 cactggccat atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt
     1681 aaagttcacg ggagacttta tctgacagca gacgtgcact ggccaggggg atcaccatcc
     1741 gtcgcccggg cgtgtcaata atatcactct gtacatccac aaacagacga taacggctct
     1801 ctcttttata ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc gtcagcaaaa
     1861 gagccgttca tttcaataaa ccgggcgacc tcagccatcc cttcctgatt ttccgctttc
     1921 cagcgttcgg cacgcagacg acgggcttca ttctgcatgg ttgtgcttac cagaccggag
     1981 atattgacat catatatgcc ttgagcaact gatagctgtc gctgtcaact gtcactgtaa
     2041 tacgctgctt catagcatac ctctttttga catacttcgg gtatacatat cagtatatat
     2101 tcttataccg caaaaatcag cgcgcaaata cgcatactgt tatctggctt ttagtaagcc
     2161 ggatccacgc gtttacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag
     2221 cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat
     2281 cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt
     2341 gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac
     2401 gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc
     2461 cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag
     2521 cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca
     2581 tatcaccagc tcaccgtctt tcattgccat acggaattcc ggatgagcat tcatcaggcg
     2641 ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa
     2701 aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa
     2761 tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat
     2821 ttttttctcc attttagctt ccttagctcc tgaaaatctc gacggatcct aactcaaaat
     2881 ccacacatta tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgcggccgc
     2941 catagtgact ggatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc
     3001 taatttaata tattgatatt tatatcattt tacgtttctc gttcagcttt tttgtacaaa
     3061 cttgtgatat catctagagc ggccgcaggt acctgaataa ctaaggccgc ttccctttag
     3121 tgagggttaa tgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca
     3181 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     3241 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     3301 caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     3361 aaaatccgat aaggatcgat tccggagcct gaatggcgaa tggacgcgcc ctgtagcggc
     3421 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3481 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3541 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3601 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3661 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3721 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3781 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3841 atattaacgc ttacaatttc gcctgtgtac cttctgaggc ggaaagaacc agctgtggaa
     3901 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3961 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     4021 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     4081 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4141 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     4201 aggctttttt ggaggcctag gcttttgcaa aaagcttgat tcttctgaca caacagtctc
     4261 gaacttaagg ctagagccac catgattgaa caagatggat tgcacgcagg ttctccggcc
     4321 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4381 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     4441 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     4501 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4561 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4621 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4681 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4741 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4801 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4861 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4921 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4981 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5041 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5101 ccgaccaagc gacgcccaac ctgccatcac gatggccgca ataaaatatc tttattttca
     5161 ttacatctgt gtgttggttt tttgtgtgaa gatccgcgta tggtgcactc tcagtacaat
     5221 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     5281 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     5341 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     5401 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     5461 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5521 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5581 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5641 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5701 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5761 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5821 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5881 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5941 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6001 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6061 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6121 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6181 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6241 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6301 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6361 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6421 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6481 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6541 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6601 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6661 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6721 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     6781 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6841 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6901 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6961 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     7021 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     7081 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     7141 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     7201 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     7261 catggctcga cagatct
//
