LOCUS       sequence-177566-        6991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Two-hybrid system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6991)
  TITLE     Rathjen lab plasmids
REFERENCE   2  (bases 1 to 6991)
  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"
COMMENT     Sequence Label: pGAD424-hL1
FEATURES             Location/Qualifiers
     source          1..6991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      15..1179
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        1184..1585
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             1631..1651
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1667..2008
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="activation domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 activation domain
                     /translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     terminator      2748..2935
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2803..2827)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(3056..4150)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(4151..4556)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4598..4614)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4598..4614)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4611..4633)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4622..4638
                     /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(4646..4652,4653..4670,4671..4676))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4691..4712
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4829..4846)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5000..5588)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5080..5099)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5759..6550,6551..6619))
                     /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     6382..6401
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6620..6724)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6792..6810
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6848..6870)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6970..6989
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gagagtgcac cataacgcat ttaagcataa acacgcacta tgccgttctt ctcatgtata
       61 tatatataca ggcaacacgc agatataggt gcgacgtgaa cagtgagctg tatgtgcgca
      121 gctcgcgttg cattttcgga agcgctcgtt ttcggaaacg ctttgaagtt cctattccga
      181 agttcctatt ctctagctag aaagtatagg aacttcagag cgcttttgaa aaccaaaagc
      241 gctctgaaga cgcactttca aaaaaccaaa aacgcaccgg actgtaacga gctactaaaa
      301 tattgcgaat accgcttcca caaacattgc tcaaaagtat ctctttgcta tatatctctg
      361 tgctatatcc ctatataacc tacccatcca cctttcgctc cttgaacttg catctaaact
      421 cgacctctac attttttatg tttatctcta gtattactct ttagacaaaa aaattgtagt
      481 aagaactatt catagagtga atcgaaaaca atacgaaaat gtaaacattt cctatacgta
      541 gtatatagag acaaaataga agaaaccgtt cataattttc tgaccaatga agaatcatca
      601 acgctatcac tttctgttca caaagtatgc gcaatccaca tcggtataga atataatcgg
      661 ggatgccttt atcttgaaaa aatgcacccg cagcttcgct agtaatcagt aaacgcggga
      721 agtggagtca ggcttttttt atggaagaga aaatagacac caaagtagcc ttcttctaac
      781 cttaacggac ctacagtgca aaaagttatc aagagactgc attatagagc gcacaaagga
      841 gaaaaaaagt aatctaagat gctttgttag aaaaatagcg ctctcgggat gcatttttgt
      901 agaacaaaaa agaagtatag attctttgtt ggtaaaatag cgctctcgcg ttgcatttct
      961 gttctgtaaa aatgcagctc agattctttg tttgaaaaat tagcgctctc gcgttgcatt
     1021 tttgttttac aaaaatgaag cacagattct tcgttggtaa aatagcgctt tcgcgttgca
     1081 tttctgttct gtaaaaatgc agctcagatt ctttgtttga aaaattagcg ctctcgcgtt
     1141 gcatttttgt tctacaaaat gaagcacaga tgcttcgttg cttgcatgca acttcttttc
     1201 tttttttttc ttttctctct cccccgttgt tgtctcacca tatccgcaat gacaaaaaaa
     1261 tgatggaaga cactaaagga aaaaattaac gacaaagaca gcaccaacag atgtcgttgt
     1321 tccagagctg atgaggggta tctcgaagca cacgaaactt tttccttcct tcattcacgc
     1381 acactactct ctaatgagca acggtatacg gccttccttc cagttacttg aatttgaaat
     1441 aaaaaaaagt ttgctgtctt gctatcaagt ataaatagac ctgcaattat taatcttttg
     1501 tttcctcgtc attgttctcg ttccctttct tccttgtttc tttttctgca caatatttca
     1561 agctatacca agcatacaat caactccaag ctttgcaaag atggataaag cggaattaat
     1621 tcccgagcct ccaaaaaaga agagaaaggt cgaattgggt accgccgcca attttaatca
     1681 aagtgggaat attgctgata gctcattgtc cttcactttc actaacagta gcaacggtcc
     1741 gaacctcata acaactcaaa caaattctca agcgctttca caaccaattg cctcctctaa
     1801 cgttcatgat aacttcatga ataatgaaat cacggctagt aaaattgatg atggtaataa
     1861 ttcaaaacca ctgtcacctg gttggacgga ccaaactgcg tataacgcgt ttggaatcac
     1921 tacagggatg tttaatacca ctacaatgga tgatgtatat aactatctat tcgatgatga
     1981 agatacccca ccaaacccaa aaaaagagat cgaattcatc aaacgcagca agggcggcaa
     2041 atactcagtg aaggataagg aggacaccca ggtggactct gaggcccgac cgatgaaaga
     2101 tgagaccttc ggcgagtaca gtgacaacga ggagaaggcc tttggcagca gccagccatc
     2161 gctcaacggg gacatcaagc ccctgggcag tgacgacagc ctggccgatt atgggggcag
     2221 cgtggatgtt cagttcaacg aggatggttc gttcattggc cagtacagtg gcaagaagga
     2281 gaaggaggcg gcagggggca atgacagctc aggggccact tcccccatca accctgcagt
     2341 agccctagaa tagcagtcga cctgcagaga tctatgaatc gtagatactg aaaaaccccg
     2401 caagttcact tcaactgtgc atcgtgcacc atctcaattt ctttcattta tacatcgttt
     2461 tgccttcttt tatgtaacta tactcctcta agtttcaatc ttggccatgt aacctctgat
     2521 ctatagaatt ttttaaatga ctagaattaa tgcccatctt ttttttggac ctaaattctt
     2581 catgaaaata tattacgagg gcttattcag aagctttgga cttcttcgcc agaggtttgg
     2641 tcaagtctcc aatcaaggtt gtcggcttgt ctaccttgcc agaaatttac gaaaagatgg
     2701 aaaagggtca aatcgttggt agatacgttg ttgacacttc taaataagcg aatttcttat
     2761 gatttatgat ttttattatt aaataagtta taaaaaaaat aagtgtatac aaattttaaa
     2821 gtgactctta ggttttaaaa cgaaaattct tattcttgag taactctttc ctgtaggtca
     2881 ggttgctttc tcaggtatag catgaggtcg ctcttattga ccacacctct accggcatgc
     2941 cggtcgaaat tcccctaccc tatgaacata ttccattttg taatttcgtg tcgtttctat
     3001 tatgaatttc atttataaag tttatgtaca aatatcataa aaaaagagaa tctttttaag
     3061 caaggatttt cttaacttct tcggcgacag catcaccgac ttcggtggta ctgttggaac
     3121 cacctaaatc accagttctg atacctgcat ccaaaacctt tttaactgca tcttcaatgg
     3181 ccttaccttc ttcaggcaag ttcaatgaca atttcaacat cattgcagca gacaagatag
     3241 tggcgatagg gttgacctta ttctttggca aatctggagc agaaccgtgg catggttcgt
     3301 acaaaccaaa tgcggtgttc ttgtctggca aagaggccaa ggacgcagat ggcaacaaac
     3361 ccaaggaacc tgggataacg gaggcttcat cggagatgat atcaccaaac atgttgctgg
     3421 tgattataat accatttagg tgggttgggt tcttaactag gatcatggcg gcagaatcaa
     3481 tcaattgatg ttgaaccttc aatgtaggaa attcgttctt gatggtttcc tccacagttt
     3541 ttctccataa tcttgaagag gccaaaacat tagctttatc caaggaccaa ataggcaatg
     3601 gtggctcatg ttgtagggcc atgaaagcgg ccattcttgt gattctttgc acttctggaa
     3661 cggtgtattg ttcactatcc caagcgacac catcaccatc gtcttccttt ctcttaccaa
     3721 agtaaatacc tcccactaat tctctgacaa caacgaagtc agtaccttta gcaaattgtg
     3781 gcttgattgg agataagtct aaaagagagt cggatgcaaa gttacatggt cttaagttgg
     3841 cgtacaattg aagttcttta cggattttta gtaaaccttg ttcaggtcta acactacctg
     3901 taccccattt aggaccaccc acagcaccta acaaaacggc atcagccttc ttggaggctt
     3961 ccagcgcctc atctggaagt ggaacacctg tagcatcgat agcagcacca ccaattaaat
     4021 gattttcgaa atcgaacttg acattggaac gaacatcaga aatagcttta agaaccttaa
     4081 tggcttcggc tgtgatttct tgaccaacgt ggtcacctgg caaaacgacg atcttcttag
     4141 gggcagacat tagaatggta tatccttgaa atatatatat atattgctga aatgtaaaag
     4201 gtaagaaaag ttagaaagta agacgattgc taaccaccta ttggaaaaaa caataggtcc
     4261 ttaaataata ttgtcaactt caagtattgt gatgcaagca tttagtcatg aacgcttctc
     4321 tattctatat gaaaagccgg ttccggcgct ctcacctttc ctttttctcc caatttttca
     4381 gttgaaaaag gtatatgcgt caggcgacct ctgaaattaa caaaaaattt ccagtcatcg
     4441 aatttgattc tgtgcgatag cgcccctgtg tgttctcgtt atgttgagga aaaaaataat
     4501 ggttgctaag agattcgaac tcttgcatct tacgatacct gagtattccc acagttgggg
     4561 gatctcgact ctagctagag gatcaattcg taatcatgtc atagctgttt cctgtgtgaa
     4621 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4681 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4741 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4801 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4861 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4921 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4981 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5041 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5101 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5161 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5221 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5281 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5341 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5401 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5461 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5521 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5581 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5641 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5701 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5761 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5821 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5881 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5941 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6001 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6061 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6121 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6181 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6241 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6301 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6361 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6421 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6481 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6541 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6601 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6661 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6721 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     6781 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     6841 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     6901 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     6961 ttaactatgc ggcatcagag cagattgtac t
//
