LOCUS       sequence-177455-        5888 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 5888)
  TITLE     Rathjen lab plasmids
REFERENCE   2  (bases 1 to 5888)
  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: pGBT9-mCALEBsplice (aa 447-566)
FEATURES             Location/Qualifiers
     source          1..5888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      13..1177
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        1182..1583
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             1611..2051
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     terminator      2463..2650
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2518..2542)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2703..3377)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(3378..3479)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(3493..3509)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3493..3509)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3506..3528)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3517..3533
                     /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(3541..3547,3548..3565,3566..3571))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3586..3607
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3724..3741)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3895..4483)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3975..3994)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4654..5445,5446..5514))
                     /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     5277..5296
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5515..5619)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5687..5705
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5743..5765)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5865..5884
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gagtgcacca taacgcattt aagcataaac acgcactatg ccgttcttct catgtatata
       61 tatatacagg caacacgcag atataggtgc gacgtgaaca gtgagctgta tgtgcgcagc
      121 tcgcgttgca ttttcggaag cgctcgtttt cggaaacgct ttgaagttcc tattccgaag
      181 ttcctattct ctagctagaa agtataggaa cttcagagcg cttttgaaaa ccaaaagcgc
      241 tctgaagacg cactttcaaa aaaccaaaaa cgcaccggac tgtaacgagc tactaaaata
      301 ttgcgaatac cgcttccaca aacattgctc aaaagtatct ctttgctata tatctctgtg
      361 ctatatccct atataaccta cccatccacc tttcgctcct tgaacttgca tctaaactcg
      421 acctctacat tttttatgtt tatctctagt attactcttt agacaaaaaa attgtagtaa
      481 gaactattca tagagtgaat cgaaaacaat acgaaaatgt aaacatttcc tatacgtagt
      541 atatagagac aaaatagaag aaaccgttca taattttctg accaatgaag aatcatcaac
      601 gctatcactt tctgttcaca aagtatgcgc aatccacatc ggtatagaat ataatcgggg
      661 atgcctttat cttgaaaaaa tgcacccgca gcttcgctag taatcagtaa acgcgggaag
      721 tggagtcagg ctttttttat ggaagagaaa atagacacca aagtagcctt cttctaacct
      781 taacggacct acagtgcaaa aagttatcaa gagactgcat tatagagcgc acaaaggaga
      841 aaaaaagtaa tctaagatgc tttgttagaa aaatagcgct ctcgggatgc atttttgtag
      901 aacaaaaaag aagtatagat tctttgttgg taaaatagcg ctctcgcgtt gcatttctgt
      961 tctgtaaaaa tgcagctcag attctttgtt tgaaaaatta gcgctctcgc gttgcatttt
     1021 tgttttacaa aaatgaagca cagattcttc gttggtaaaa tagcgctttc gcgttgcatt
     1081 tctgttctgt aaaaatgcag ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc
     1141 atttttgttc tacaaaatga agcacagatg cttcgttgct tgcatgcaac ttcttttctt
     1201 tttttttctt ttctctctcc cccgttgttg tctcaccata tccgcaatga caaaaaaatg
     1261 atggaagaca ctaaaggaaa aaattaacga caaagacagc accaacagat gtcgttgttc
     1321 cagagctgat gaggggtatc tcgaagcaca cgaaactttt tccttccttc attcacgcac
     1381 actactctct aatgagcaac ggtatacggc cttccttcca gttacttgaa tttgaaataa
     1441 aaaaaagttt gctgtcttgc tatcaagtat aaatagacct gcaattatta atcttttgtt
     1501 tcctcgtcat tgttctcgtt ccctttcttc cttgtttctt tttctgcaca atatttcaag
     1561 ctataccaag catacaatca actccaagct tgaagcaagc ctcctgaaag atgaagctac
     1621 tgtcttctat cgaacaagca tgcgatattt gccgacttaa aaagctcaag tgctccaaag
     1681 aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac tctcccaaaa
     1741 ccaaaaggtc tccgctgact agggcacatc tgacagaagt ggaatcaagg ctagaaagac
     1801 tggaacagct atttctactg atttttcctc gagaagacct tgacatgatt ttgaaaatgg
     1861 attctttaca ggatataaaa gcattgttaa caggattatt tgtacaagat aatgtgaata
     1921 aagatgccgt cacagataga ttggcttcag tggagactga tatgcctcta acattgagac
     1981 agcatagaat aagtgcgaca tcatcatcgg aagagagtag taacaaaggt caaagacagt
     2041 tgactgtatc gccggaattc ctctatctgc tcaagactga gaataccaag ctgcggagga
     2101 ccaataaatt ccggacccca tctgagctcc acaacgacaa cttctccctc tccaccattg
     2161 ccgagggctc tcatccaaat gtaaggaaat tttgcgacac tccccgtgtc tcctcccccc
     2221 atgcccgtgc cttggctcac tatgataaca ttgtctgtca ggacgacccc agcgctcccc
     2281 acaaaatcca ggaccctctc aagtcccgcc tgaaggagga agagtccttt aacatccaga
     2341 actccatgtc acccaaactt gagggtggca aaggtgacca ggatgacttg ggggtgaact
     2401 gtctgcagaa taacctaacc tgagactgag gatccgtcga cctgcagcca agctaattcc
     2461 gggcgaattt cttatgattt atgattttta ttattaaata agttataaaa aaaataagtg
     2521 tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa attcttattc ttgagtaact
     2581 ctttcctgta ggtcaggttg ctttctcagg tatagcatga ggtcgctctt attgaccaca
     2641 cctctaccgg catgccggca agtgcacaaa caatacttaa ataaatacta ctcagtaata
     2701 acctatttct tagcattttt gacgaaattt gctattttgt tagagtcttt tacaccattt
     2761 gtctccacac ctccgcttac atcaacacca ataacgccat ttaatctaag cgcatcacca
     2821 acattttctg gcgtcagtcc accagctaac ataaaatgta agctttcggg gctctcttgc
     2881 cttccaaccc agtcagaaat cgagttccaa tccaaaagtt cacctgtccc acctgcttct
     2941 gaatcaaaca agggaataaa cgaatgaggt ttctgtgaag ctgcactgag tagtatgttg
     3001 cagtcttttg gaaatacgag tcttttaata actggcaaac cgaggaactc ttggtattct
     3061 tgccacgact catctccatg cagttggacg atatcaatgc cgtaatcatt gaccagagcc
     3121 aaaacatcct ccttaggttg attacgaaac acgccaacca agtatttcgg agtgcctgaa
     3181 ctatttttat atgcttttac aagacttgaa attttccttg caataaccgg gtcaattgtt
     3241 ctctttctat tgggcacaca tataataccc agcaagtcag catcggaatc tagagcacat
     3301 tctgcggcct ctgtgctctg caagccgcaa actttcacca atggaccaga actacctgtg
     3361 aaattaataa cagacatact ccaagctgcc tttgtgtgct taatcacgta tactcacgtg
     3421 ctcaatagtc accaatgccc tccctcttgg ccctctcctt ttcttttttc gaccgaatta
     3481 attcgtaatc atgtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3541 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3601 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3661 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3721 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3781 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3841 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3901 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3961 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4021 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4081 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     4141 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4201 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4261 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4321 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4381 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4441 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4501 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4561 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4621 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4681 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4741 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4801 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4861 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4921 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4981 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     5041 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     5101 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     5161 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     5221 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     5281 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5341 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5401 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5461 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5521 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5581 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5641 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     5701 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     5761 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     5821 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     5881 tgtactga
//
