LOCUS       sequence-12162-e        7279 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 7279)
  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 7279)
  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-Gate2
FEATURES             Location/Qualifiers
     source          1..7279
                     /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    1362..1486
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1511..1516,1517..1534,1535..1541)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1595..2254
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1661..1680)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2596..2901
                     /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     2780..2799
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2942..3066)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3151..3272
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3162..3181)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3216..3235
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3406..3861
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3493..3512)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3703..3724
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3876..4233
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3876..4067
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3899..3919)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4084..4219
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4284..5078
                     /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(4338..4357)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4948..4967
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5142..5190
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5217..5236)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5336..5358
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5396..5414)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5482..5586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5587..5655,5656..6447)
                     /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(5805..5824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6618..7206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7107..7126
                     /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 aacaagtttg tacaaaaaag
     1381 ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa
     1441 aacagactac ataatactgt aaaacacaac atatccagtc actatggcgg ccgcattagg
     1501 caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggattt tgagttagga
     1561 tccgtcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
     1621 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc
     1681 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa
     1741 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
     1801 tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca
     1861 cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata
     1921 ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga
     1981 aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc
     2041 ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc
     2101 cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat
     2161 tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca
     2221 acagtactgc gatgagtggc agggcggggc gtaaagatct ggatccggct tactaaaagc
     2281 cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat
     2341 gtatacccga agtatgtcaa aaagaggtat gctatgaagc agcgtattac agtgacagtt
     2401 gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa
     2461 gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc
     2521 aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga
     2581 acaggggctg gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt
     2641 ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc
     2701 ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat
     2761 atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt
     2821 atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac
     2881 ctgatgttct ggggaatata aatgtcaggc tcccttatac acagccagtc tgcaggtcga
     2941 ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat
     3001 ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt tcttgtacaa
     3061 agtggttgat atcatctaga gcggccgcag gtacctgaat aactaaggcc gcttcccttt
     3121 agtgagggtt aatgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca
     3181 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     3241 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     3301 ttcaggttca gggggagatg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     3361 gtaaaatccg ataaggatcg attccggagc ctgaatggcg aatggacgcg ccctgtagcg
     3421 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3481 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3541 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3601 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3661 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3721 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3781 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3841 aaatattaac gcttacaatt tcgcctgtgt accttctgag gcggaaagaa ccagctgtgg
     3901 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3961 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     4021 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     4081 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4141 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     4201 ggaggctttt ttggaggcct aggcttttgc aaaaagcttg attcttctga cacaacagtc
     4261 tcgaacttaa ggctagagcc accatgattg aacaagatgg attgcacgca ggttctccgg
     4321 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     4381 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     4441 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     4501 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     4561 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     4621 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     4681 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     4741 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     4801 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     4861 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     4921 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     4981 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     5041 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     5101 gaccgaccaa gcgacgccca acctgccatc acgatggccg caataaaata tctttatttt
     5161 cattacatct gtgtgttggt tttttgtgtg aagatccgcg tatggtgcac tctcagtaca
     5221 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     5281 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5341 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     5401 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     5461 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5521 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5581 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5641 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5701 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5761 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5821 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5881 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5941 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     6001 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     6061 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     6121 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     6181 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6241 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6301 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6361 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6421 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6481 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6541 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6601 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6661 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6721 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6781 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6841 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6901 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6961 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     7021 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     7081 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     7141 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7201 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7261 cacatggctc gacagatct
//
