LOCUS       sequence-177484-        8615 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 8615)
  TITLE     Rathjen lab plasmids
REFERENCE   2  (bases 1 to 8615)
  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: pAS2-mCALEBcyt (aa 465-539)
FEATURES             Location/Qualifiers
     source          1..8615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1005..1709
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             1737..2177
                     /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      2496..2683
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2551..2575)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2707..2723)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2707..2723)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2720..2742)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2731..2747
                     /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(2755..2761,2762..2779,2780..2785))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2800..2821
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2938..2955)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3109..3697)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3189..3208)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3868..4659,4660..4728))
                     /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     4491..4510
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4729..4833)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4860..6202
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5823..5870)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     6243..6261
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6299..6321)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6421..6440
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6461..6742
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             6743..7417
                     /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"
     rep_origin      complement(7520..7975)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7657..7678)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7869..7888
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8101..8123
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8115..8132
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8116..8132
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      complement(8166..8413)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     8379..8397
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 gatagaaata atacagaagt agatgttgaa ttagattaaa ctgaagatat ataatttatt
       61 ggaaaataca tagagttttt gttgatgcgc ttaagcgatc aattcaacaa caccaccagc
      121 agctctgatt ttttcttcag ccaacttgga gacgaatcta gctttgacga taactggaac
      181 atttgggatt ctacccttac ccaagatctt accgtaaccg gctgccaaag tgtcaataac
      241 tggagcagtt tccttagaag cagatttcaa gtattggtct ctcttgtctt ctgggatcaa
      301 tgtccacaat ttgtccaagt tcaagactgg cttccagaaa tgagcttgtt gcttgtggaa
      361 gtatctcata ccaaccttac cgaaataacc tggatggtat ttatccatgt taattctgtg
      421 gtgatgttga ccaccggcca tacctctacc accggggtgc tttctgtgct taccgatacg
      481 acctttaccg gctgagacgt gacctctgtg ctttctagtc ttagtgaatc tggaaggcat
      541 tcttgattag ttggatgatt gttctgggat ttaatgcaaa aaaatcacta agaaggaaaa
      601 aaatcaacgg agaaagcaaa cgccatctta aatatacggg atacagatga aaggtttgaa
      661 cctatctggg aaaatacgca ttaaacaagc gaaaaactgc gaggaaaatt gtttgcgtct
      721 ctgcgggcta ttcacgcgcc agaggaaaaa taggaaaaat aacaggcatt agaaaaataa
      781 ttttgatttt ggtaatgtgt gggtccctgg tgtacagatg ttacattggt tacagtactc
      841 ttgtttttgc tgtgtttttc gatgaatctc caaaatggtt gttagcacat ggaagagtca
      901 ccgatgctaa gttatctcta tgtaagctac gtggcgtgac ttttgatgaa gccgcacaag
      961 agatacagga ttggcaactg caaatagaat ctggggatct agatatcctt ttgttgtttc
     1021 cgggtgtaca atatggactt cctcttttct ggcaaccaaa cccatacatc gggattccta
     1081 taataccttc gttggtctcc ctaacatgta ggtggcggag gggagatata caatagaaca
     1141 gataccagac aagacataat gggctaaaca agactacacc aattacactg cctcattgat
     1201 ggtggtacat aacgaactaa tactgtagcc ctagacttga tagccatcat catatcgaag
     1261 tttcactacc ctttttccat ttgccatcta ttgaagtaat aataggcgca tgcaacttct
     1321 tttctttttt tttcttttct ctctcccccg ttgttgtctc accatatccg caatgacaaa
     1381 aaaatgatgg aagacactaa aggaaaaaat taacgacaaa gacagcacca acagatgtcg
     1441 ttgttccaga gctgatgagg ggtatctcga agcacacgaa actttttcct tccttcattc
     1501 acgcacacta ctctctaatg agcaacggta tacggccttc cttccagtta cttgaatttg
     1561 aaataaaaaa aagtttgctg tcttgctatc aagtataaat agacctgcaa ttattaatct
     1621 tttgtttcct cgtcattgtt ctcgttccct ttcttccttg tttctttttc tgcacaatat
     1681 ttcaagctat accaagcata caatcaactc caagcttgaa gcaagcctcc tgaaagatga
     1741 agctactgtc ttctatcgaa caagcatgcg atatttgccg acttaaaaag ctcaagtgct
     1801 ccaaagaaaa accgaagtgc gccaagtgtc tgaagaacaa ctgggagtgt cgctactctc
     1861 ccaaaaccaa aaggtctccg ctgactaggg cacatctgac agaagtggaa tcaaggctag
     1921 aaagactgga acagctattt ctactgattt ttcctcgaga agaccttgac atgattttga
     1981 aaatggattc tttacaggat ataaaagcat tgttaacagg attatttgta caagataatg
     2041 tgaataaaga tgccgtcaca gatagattgg cttcagtgga gactgatatg cctctaacat
     2101 tgagacagca tagaataagt gcgacatcat catcggaaga gagtagtaac aaaggtcaaa
     2161 gacagttgac tgtatcgccg gtattgcaat acccagcttt gactcatatg gccatggagg
     2221 ccgaattcac cccatctgag ctccacaacg acaacttctc cctctccacc attgccgagg
     2281 gctctcatcc aaatgacgac cccagcgctc cccacaaaat ccaggaccct ctcaagtccc
     2341 gcctgaagga ggaagagtcc tttaacatcc agaactccat gtcacccaaa cttgagggtg
     2401 gcaaaggtga ccaggatgac ttgggggtga actgtctgca gaataaccta acctgagact
     2461 gaggatccgt cgacctgcag ccaagctaat tccgggcgaa tttcttatga tttatgattt
     2521 ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg
     2581 ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc
     2641 aggtatagca tgaggtcgct cttattgacc acacctctac cggcatgcaa gcttggcgta
     2701 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2761 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     2821 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     2881 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     2941 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3001 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3061 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3121 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3181 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3241 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3301 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3361 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3421 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactagta acaggattag
     3481 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3541 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3601 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3661 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3721 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3781 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3841 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3901 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3961 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4021 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4081 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4141 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4201 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4261 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4321 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4381 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4441 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4501 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4561 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4621 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4681 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4741 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4801 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     4861 acgaagcatc tgtgcttcat tttgtagaac aaaaatgcaa cgcgagagcg ctaatttttc
     4921 aaacaaagaa tctgagctgc atttttacag aacagaaatg caacgcgaaa gcgctatttt
     4981 accaacgaag aatctgtgct tcatttttgt aaaacaaaaa tgcaacgcga gagcgctaat
     5041 ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg cgagagcgct
     5101 attttaccaa caaagaatct atacttcttt tttgttctac aaaaatgcat cccgagagcg
     5161 ctatttttct aacaaagcat cttagattac tttttttctc ctttgtgcgc tctataatgc
     5221 agtctcttga taactttttg cactgtaggt ccgttaaggt tagaagaagg ctactttggt
     5281 gtctattttc tcttccataa aaaaagcctg actccacttc ccgcgtttac tgattactag
     5341 cgaagctgcg ggtgcatttt ttcaagataa aggcatcccc gattatattc tataccgatg
     5401 tggattgcgc atactttgtg aacagaaagt gatagcgttg atgattcttc attggtcaga
     5461 aaattatgaa cggtttcttc tattttgtct ctatatacta cgtataggaa atgtttacat
     5521 tttcgtattg ttttcgattc actctatgaa tagttcttac tacaattttt ttgtctaaag
     5581 agtaatacta gagataaaca taaaaaatgt agaggtcgag tttagatgca agttcaagga
     5641 gcgaaaggtg gatgggtagg ttatataggg atatagcaca gagatatata gcaaagagat
     5701 acttttgagc aatgtttgtg gaagcggtat tcgcaatatt ttagtagctc gttacagtcc
     5761 ggtgcgtttt tggttttttg aaagtgcgtc ttcagagcgc ttttggtttt caaaagcgct
     5821 ctgaagttcc tatactttct agagaatagg aacttcggaa taggaacttc aaagcgtttc
     5881 cgaaaacgag cgcttccgaa aatgcaacgc gagctgcgca catacagctc actgttcacg
     5941 tcgcacctat atctgcgtgt tgcctgtata tatatataca tgagaagaac ggcatagtgc
     6001 gtgtttatgc ttaaatgcgt acttatatgc gtctatttat gtaggatgaa aggtagtcta
     6061 gtacctcctg tgatattatc ccattccatg cggggtatcg tatgcttcct tcagcactac
     6121 cctttagctg ttctatatgc tgccactcct caattggatt agtctcatcc ttcaatgcta
     6181 tcatttcctt tgatattgga tcatactaag aaaccattat tatcatgaca ttaacctata
     6241 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
     6301 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     6361 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
     6421 cggcatcaga gcagattgta ctgagagtgc accatagatc aacgacatta ctatatatat
     6481 aatataggaa gcatttaata gaacagcatc gtaatatatg tgtactttgc agttatgacg
     6541 ccagatggca gtagtggaag atattcttta ttgaaaaata gcttgtcacc ttacgtacaa
     6601 tcttgatccg gagcttttct ttttttgccg attaagaatt aattcggtcg aaaaaagaaa
     6661 aggagagggc caagagggag ggcattggtg actattgagc acgtgagtat acgtgattaa
     6721 gcacacaaag gcagcttgga gtatgtctgt tattaatttc acaggtagtt ctggtccatt
     6781 ggtgaaagtt tgcggcttgc agagcacaga ggccgcagaa tgtgctctag attccgatgc
     6841 tgacttgctg ggtattatat gtgtgcccaa tagaaagaga acaattgacc cggttattgc
     6901 aaggaaaatt tcaagtcttg taaaagcata taaaaatagt tcaggcactc cgaaatactt
     6961 ggttggcgtg tttcgtaatc aacctaagga ggatgttttg gctctggtca atgattacgg
     7021 cattgatatc gtccaactgc atggagatga gtcgtggcaa gaataccaag agttcctcgg
     7081 tttgccagtt attaaaagac tcgtatttcc aaaagactgc aacatactac tcagtgcagc
     7141 ttcacagaaa cctcattcgt ttattccctt gtttgattca gaagcaggtg ggacaggtga
     7201 acttttggat tggaactcga tttctgactg ggttggaagg caagagagcc ccgaaagctt
     7261 acattttatg ttagctggtg gactgacgcc agaaaatgtt ggtgatgcgc ttagattaaa
     7321 tggcgttatt ggtgttgatg taagcggagg tgtggagaca aatggtgtaa aagactctaa
     7381 caaaatagca aatttcgtca aaaatgctaa gaaataggtt attactgagt agtatttatt
     7441 taagtattgt ttgtgcactt gccgatctat gcggtgtgaa ataccgcaca gatgcgtaag
     7501 gagaaaatac cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     7561 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     7621 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     7681 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     7741 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     7801 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     7861 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     7921 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt
     7981 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     8041 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     8101 cccagtcacg acgttgtaaa acgacggcca gtgccaagct ttcgcgagct cgagatcccg
     8161 agcttgcaaa ttaaagcctt cgagcgtccc aaaaccttct caagcaaggt tttcagtata
     8221 atgttacatg cgtacacgcg tctgtacaga aaaaaaagaa aaatttgaaa tataaataac
     8281 gttcttaata ctaacataac tataaaaaaa taaataggga cctagacttc aggttgtcta
     8341 actccttcct tttcggttag agcggatgtg gggggagggc gtgaatgtaa gcgtgacata
     8401 actaattaca tgatatcgac aaaggaaaag gggcctgttt actcacaggc ttttttcaag
     8461 taggtaatta agtcgtttct gtctttttcc ttcttcaacc caccaaaggc catcttggta
     8521 cttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
     8581 tttttttttt tttttttttt tttttttttt ttttt
//
