LOCUS       sequence-9376-ex        9030 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 9030)
  AUTHORS   Chen G, DenBoer L, Shin J
  TITLE     Design of a single plasmid-based modified yeast one-hybrid system 
            for investigation of in vivo protein-protein and protein-DNA 
            interactions.
  JOURNAL   Biotechniques. 2008 Sep . 45(3):295-304.
  PUBMED    18778253
REFERENCE   2  (bases 1 to 9030)
  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"; journalName: 
            "Biotechniques. 2008 Sep . 45(3):295-304."
COMMENT     Sequence Label: pCETF
FEATURES             Location/Qualifiers
     source          1..9030
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..406
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             452..472
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             488..829
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="activation domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 activation domain
                     /translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     promoter        835..853
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             875..904
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     terminator      1328..1515
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     CDS             complement(1633..2727)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(2728..3132)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(3174..3190)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3174..3190)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3187..3209)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3198..3214
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3222..3228,3229..3246,3247..3252))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3405..3422)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3576..4164)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3656..3675)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4335..5126,5127..5195))
                     /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     4958..4977
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5196..5300)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(5343..5530)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     CDS             complement(5978..6004)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      6007..6016
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6023..6041)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(6074..6094)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(6140..6845)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     primer_bind     7652..7670
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7708..7730)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7830..7849
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      7866..9030
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 gcttgcatgc aacttctttt cttttttttt cttttctctc tcccccgttg ttgtctcacc
       61 atatccgcaa tgacaaaaaa aatgatggaa gacactaaag gaaaaaatta acgacaaaga
      121 cagcaccaac agatgtcgtt gttccagagc tgatgagggg tatcttcgaa cacacgaaac
      181 tttttccttc cttcattcac gcacactact ctctaatgag caacggtata cggccttcct
      241 tccagttact tgaatttgaa ataaaaaaag tttgccgctt tgctatcaag tataaataga
      301 cctgcaatta ttaatctttt gtttcctcgt cattgttctc gttccctttc ttccttgttt
      361 ctttttctgc acaatatttc aagctatacc aagcatacaa tcaactccaa gctttgcaaa
      421 gatggataaa gcggaattaa ttcccgagcc tccaaaaaag aagagaaagg tcgaattggg
      481 taccgccgcc aattttaatc aaagtgggaa tattgctgat agctcattgt ccttcacttt
      541 cactaacagt agcaacggtc cgaacctcat aacaactcaa acaaattctc aagcgctttc
      601 acaaccaatt gcctcctcta acgttcatga taacttcatg aataatgaaa tcacggctag
      661 taaaattgat gatggtaata attcaaaacc actgtcacct ggttggacgg accaaactgc
      721 gtataacgcg tttggaatca ctacagggat gtttaatacc actacaatgg atgatgtata
      781 taactatcta ttcgatgatg aagatacccc accaaaccca aaaaaagaga tctttaatac
      841 gactcactat agggcgagcg ccgccatcat ggaggagcag aagctgatct cagaggagga
      901 cctgcatatg gccatggagg ccgcggtcga caaggtgcgc gctctagatg atcatgaatc
      961 gtagatactg aaaaaccccg caagttcact tcaactgtgc atcgtgcacc atctcaattt
     1021 ctttcattta tacatcgttt tgccttcttt tatgtaacta tactcctcta agtttcaatc
     1081 ttggccatgt aacctctgat ctatagaatt ttttaaatga ctagaattaa tgcccatctt
     1141 ttttttggac ctaaattctt catgaaaata tattacgagg gcttattcag aagctttgga
     1201 cttcttcgcc agaggtttgg tcaagtctcc aatcaaggtt gtcggcttgt ctaccttgcc
     1261 agaaatttac gaaaagatgg aaaagggtca aatcgttggt agatacgttg ttgacacttc
     1321 taaataagcg aatttcttat gatttatgat ttttattatt aaataagtta taaaaaaaat
     1381 aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct tgttcttgag
     1441 taactctttc ctgtaggtca ggttgctttc tcaggtatag catgaggtcg ctcttattga
     1501 ccacacctct accggcatgc ccgaaattcc cctaccctat gaacatattc cattttgtaa
     1561 tttcgtgtcg tttctattat gaatttcatt tataaagttt atgtacaaat atcataaaaa
     1621 aagagaatct ttttaagcaa ggattttctt aacttcttcg gcgacagcat caccgacttc
     1681 ggtggtactg ttggaaccac ctaaatcacc agttctgata cctgcatcca aaaccttttt
     1741 aactgcatct tcaatggcct taccttcttc aggcaagttc aatgacaatt tcaacatcat
     1801 tgcagcagac aagatagtgg cgatagggtc aaccttattc tttggcaaat ctggagcaga
     1861 accgtggcat ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag aggccaagga
     1921 cgcagatggc aacaaaccca aggaacctgg gataacggag gcttcatcgg agatgatatc
     1981 accaaacatg ttgctggtga ttataatacc atttaggtgg gttgggttct taactaggat
     2041 catggcggca gaatcaatca attgatgttg aaccttcaat gtaggaaatt cgttcttgat
     2101 ggtttcctcc acagtttttc tccataatct tgaagaggcc aaaacattag ctttatccaa
     2161 ggaccaaata ggcaatggtg gctcatgttg tagggccatg aaagcggcca ttcttgtgat
     2221 tctttgcact tctggaacgg tgtattgttc actatcccaa gcgacaccat caccatcgtc
     2281 ttcctttctc ttaccaaagt aaatacctcc cactaattct ctgacaacaa cgaagtcagt
     2341 acctttagca aattgtggct tgattggaga taagtctaaa agagagtcgg atgcaaagtt
     2401 acatggtctt aagttggcgt acaattgaag ttctttacgg atttttagta aaccttgttc
     2461 aggtctaaca ctacctgtac cccatttagg accacccaca gcacctaaca aaacggcatc
     2521 aaccttcttg gaggcttcca gcgcctcatc tggaagtggg acacctgtag catcgatagc
     2581 agcaccacca attaaatgat tttcgaaatc gaacttgaca ttggaacgaa catcagaaat
     2641 agctttaaga accttaatgg cttcggctgt gatttcttga ccaacgtggt cacctggcaa
     2701 aacgacgatc ttcttagggg cagacattag aatggtatat ccttgaaata tatatatata
     2761 ttgctgaaat gtaaaaggta agaaaagtta gaaagtaaga cgattgctaa ccacctattg
     2821 gaaaaaacaa taggtcctta aataatattg tcaacttcaa gtattgtgat gcaagcattt
     2881 agtcatgaac gcttctctat tctatatgaa aagccggttc cggcctctca cctttccttt
     2941 ttctcccaat ttttcagttg aaaaaggtat atgcgtcagg cgacctctga aattaacaaa
     3001 aaatttccag tcatcgaatt tgattctgtg cgatagcgcc cctgtgtgtt ctcgttatgt
     3061 tgaggaaaaa aataatggtt gctaagagat tcgaactctt gcatcttacg atacctgagt
     3121 attcccacag ttggggatct cgactctagc tagaggatca attcgtaatc atggtcatag
     3181 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     3241 ataaagtgta aagcctgggg tgcctaatga gtgaggtaac tcacattaat tgcgttgcgc
     3301 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tggattaatg aatcggccaa
     3361 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     3421 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3481 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3541 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3601 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3661 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3721 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3781 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3841 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3901 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     3961 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     4021 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4081 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4141 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4201 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4261 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4321 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4381 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4441 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4501 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4561 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4621 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     4681 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4741 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4801 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4861 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4921 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     4981 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5041 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5101 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5161 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5221 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5281 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt caagcttgca
     5341 tgccggtaga ggtgtggtca ataagagcga cctcatgcta tacctgagaa agcaacctga
     5401 cctacaggaa agagttactc aagaataaga attttcgttt taaaacctaa gagtcacttt
     5461 aaaatttgta tacacttatt ttttttataa cttatttaat aataaaaatc ataaatcata
     5521 agaaattcgc ttatttagaa gtgtcaacaa cgtatctacc aacgatttga cccttttcca
     5581 tcttttcgta aatttctggc aaggtagaca agccgacaac cttgattgga gacttgacca
     5641 aacctctggc gaagaagtcc aaagcttctg aataagccct cgtaatatat tttcatgaag
     5701 aatttaggtc caaaaaaaag atgggcatta attctagtca tttaaaaaat tctatagatc
     5761 agaggttaca tggccaagat tgaaacttag aggagtatag ttacataaaa gaaggcaaaa
     5821 cgatgtataa atgaaagaaa ttgagatggt gcacgatgca cagttgaagt gaacttgcgg
     5881 ggtttttcag tatctacgat tcatctgcag ctcgagctcg atggatcccg tatcgatgcc
     5941 cacccgggtg gaattcactg gcctccatgg ccatatgagc gtaatctggt acgtcgtatg
     6001 ggtactccat ggcggcgctc gccctatagt gagtcgtatt aaagatctct ttattggcgg
     6061 cggtacccaa ttcgaccttt ctcttctttt ttggaggctc gggaattaat tccgctttat
     6121 ccatctttgc aaagcttgga gttgattgta tgcttggtat agcttgaaat attgtgcaga
     6181 aaaagaaaca aggaagaaag ggaacgagaa caatgacgag gaaacaaaag attaataatt
     6241 gcaggtctat ttatacttga tagcaagaca gcaaactttt ttttatttca aattcaagta
     6301 actggaagga aggccgtata ccgttgctca ttagagagta gtgtgcgtga atgaaggaag
     6361 gaaaaagttt cgtgtgcttc gagatacccc tcatcagctc tggaacaacg acatctgttg
     6421 gtgctgtctt tgtcgttaat tttttccttt agtgtcttcc atcatttttt ttgtcattgc
     6481 ggatatggtg agacaacaac gggggagaga gaaaagaaaa aaaaagaaaa gaagttgcat
     6541 gcgcctatta ttacttcaat agatggcaaa tggaaaaagg gtagtgaaac ttcgatatga
     6601 tgatggctat caagtctagg gctacagtat tagttcgtta tgtaccacca tcaatgaggc
     6661 agtgtaattg gtgtagtctt gtttagccca ttatgtcttg tctggtatct gttctattgt
     6721 atatctcccc tccgccacct acatgttagg gagaccaacg aaggtattat aggaatcccg
     6781 atgtatgggt ttggttgcca gaaaagagga agtccatatt gtacacccgg aaacaacaaa
     6841 aggatatccg aaatattcca cggtttagaa aaaaatcgga aaagagcgcg gaggggtgtt
     6901 accccccttc tctactagca ttggacttta attaatatat gtgcatagga gaagtgtaaa
     6961 gttcccttcc atattgtaac ataataaagt gcacacccaa atgaattgaa agcgtactca
     7021 aacagacaac catttccagt gttgtatgta cctgtctatt tatactggta gcaaccctat
     7081 tgctgtttcc tcttcaaagt actctagcgg ttatgcgcgt ctcaccttca aggtcatggt
     7141 cgctctattg ttcgcaccac cggcaaactc gcgtctcgca agtcttggct cattcttcta
     7201 gtatactcat gttgcaaatg cactcaggtt ctttcggcaa cttaaataat gacaccagtt
     7261 gtcgtggtcg tcatcatcgc aaccccaacc ggcattctta ttgcttctcc aatctcgccc
     7321 cttagcgcag ggtaaacctt ggaaaatgca ggcgcaaaaa actccgccgg gcacagcctc
     7381 acgcccagcg ttatcgccgg gccggcaaga gcgcgggtcc gccacagagt cagcatgatt
     7441 gtgcaattgc gtaaactcgt tttttcggcg ccgcaaagcc aaatacatca tatcaacact
     7501 tttcacttta tttttcgttc gacccttata tttgtctttt gccttcatgc tccttgattt
     7561 cctatttcat ttaccatcat ttcttcgatc ccggatctcg acctgcaggg acgtctaaga
     7621 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     7681 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     7741 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     7801 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     7861 ccataacgca tttaagcata aacacgcact atgccgttct tctcatgtat atatatatac
     7921 aggcaacacg cagatatagg tgcgacgtga acagtgagct gtatgtgcgc agctcgcgtt
     7981 gcattttcgg aagcgctcgt tttcggaaac gctttgaagt tcctattccg aagttcctat
     8041 tctctagcta gaaagtatag gaacttcaga gcgcttttga aaaccaaaag cgctctgaag
     8101 acgcactttc aaaaaaccaa aaacgcaccg gactgtaacg agctactaaa atattgcgaa
     8161 taccgcttcc acaaacattg ctcaaaagta tctctttgct atatatctct gtgctatatc
     8221 cctatataac ctacccatcc acctttcgct ccttgaactt gcatctaaac tcgacctcta
     8281 cattttttat gtttatctct agtattactc tttagacaaa aaaattgtag taagaactat
     8341 tcatagagtg aatcgaaaac aatacgaaaa tgtaaacatt tcctatacgt agtatataga
     8401 gacaaaatag aagaaaccgt tcataatttt ctgaccaatg aagaatcatc aacgctatca
     8461 ctttctgttc acaaagtatg cgcaatccac atcggtatag aatataatcg gggatgcctt
     8521 tatcttgaaa aaatgcaccc gcagcttcgc tagtaatcag taaacgcggg aagtggagtc
     8581 aggctttttt tatggaagag aaaatagaca ccaaagtagc cttcttctaa ccttaacgga
     8641 cctacagtgc aaaaagttat caagagactg cattatagag cgcacaaagg agaaaaaaag
     8701 taatctaaga tgctttgtta gaaaaatagc gctctcggga tgcatttttg tagaacaaaa
     8761 aagaagtata gattctttgt tggtaaaata gcgctctcgc gttgcatttc tgttctgtaa
     8821 aaatgcagct cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat ttttgtttta
     8881 caaaaatgaa gcacagattc ttcgttggta aaatagcgct ttcgcgttgc atttctgttc
     8941 tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt tgcatttttg
     9001 ttctacaaaa tgaagcacag atgcttcgtt
//
