LOCUS       sequence-177477-        8473 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 8473)
  TITLE     Rathjen lab plasmids
REFERENCE   2  (bases 1 to 8473)
  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-chCALEBcyt (aa 522-551)
FEATURES             Location/Qualifiers
     source          1..8473
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1004..1708
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             1736..2176
                     /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      2354..2541
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2409..2433)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2565..2581)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2565..2581)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2578..2600)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2589..2605
                     /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(2613..2619,2620..2637,2638..2643))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2658..2679
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2796..2813)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2967..3555)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3047..3066)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3726..4517,4518..4586))
                     /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     4349..4368
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4587..4691)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4718..6060
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5681..5728)
                     /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     6101..6119
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6157..6179)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6279..6298
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6319..6600
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             6601..7275
                     /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(7378..7833)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7515..7536)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7727..7746
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7959..7981
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7973..7990
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7974..7990
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      complement(8024..8271)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     8237..8255
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 atagaaataa tacagaagta gatgttgaat tagattaaac tgaagatata taatttattg
       61 gaaaatacat agagtttttg ttgatgcgct taagcgatca attcaacaac accaccagca
      121 gctctgattt tttcttcagc caacttggag acgaatctag ctttgacgat aactggaaca
      181 tttgggattc tacccttacc caagatctta ccgtaaccgg ctgccaaagt gtcaataact
      241 ggagcagttt ccttagaagc agatttcaag tattggtctc tcttgtcttc tgggatcaat
      301 gtccacaatt tgtccaagtt caagactggc ttccagaaat gagcttgttg cttgtggaag
      361 tatctcatac caaccttacc gaaataacct ggatggtatt tatccatgtt aattctgtgg
      421 tgatgttgac caccggccat acctctacca ccggggtgct ttctgtgctt accgatacga
      481 cctttaccgg ctgagacgtg acctctgtgc tttctagtct tagtgaatct ggaaggcatt
      541 cttgattagt tggatgattg ttctgggatt taatgcaaaa aaatcactaa gaaggaaaaa
      601 aatcaacgga gaaagcaaac gccatcttaa atatacggga tacagatgaa aggtttgaac
      661 ctatctggga aaatacgcat taaacaagcg aaaaactgcg aggaaaattg tttgcgtctc
      721 tgcgggctat tcacgcgcca gaggaaaaat aggaaaaata acaggcatta gaaaaataat
      781 tttgattttg gtaatgtgtg ggtccctggt gtacagatgt tacattggtt acagtactct
      841 tgtttttgct gtgtttttcg atgaatctcc aaaatggttg ttagcacatg gaagagtcac
      901 cgatgctaag ttatctctat gtaagctacg tggcgtgact tttgatgaag ccgcacaaga
      961 gatacaggat tggcaactgc aaatagaatc tggggatcta gatatccttt tgttgtttcc
     1021 gggtgtacaa tatggacttc ctcttttctg gcaaccaaac ccatacatcg ggattcctat
     1081 aataccttcg ttggtctccc taacatgtag gtggcggagg ggagatatac aatagaacag
     1141 ataccagaca agacataatg ggctaaacaa gactacacca attacactgc ctcattgatg
     1201 gtggtacata acgaactaat actgtagccc tagacttgat agccatcatc atatcgaagt
     1261 ttcactaccc tttttccatt tgccatctat tgaagtaata ataggcgcat gcaacttctt
     1321 ttcttttttt ttcttttctc tctcccccgt tgttgtctca ccatatccgc aatgacaaaa
     1381 aaatgatgga agacactaaa ggaaaaaatt aacgacaaag acagcaccaa cagatgtcgt
     1441 tgttccagag ctgatgaggg gtatctcgaa gcacacgaaa ctttttcctt ccttcattca
     1501 cgcacactac tctctaatga gcaacggtat acggccttcc ttccagttac ttgaatttga
     1561 aataaaaaaa agtttgctgt cttgctatca agtataaata gacctgcaat tattaatctt
     1621 ttgtttcctc gtcattgttc tcgttccctt tcttccttgt ttctttttct gcacaatatt
     1681 tcaagctata ccaagcatac aatcaactcc aagcttgaag caagcctcct gaaagatgaa
     1741 gctactgtct tctatcgaac aagcatgcga tatttgccga cttaaaaagc tcaagtgctc
     1801 caaagaaaaa ccgaagtgcg ccaagtgtct gaagaacaac tgggagtgtc gctactctcc
     1861 caaaaccaaa aggtctccgc tgactagggc acatctgaca gaagtggaat caaggctaga
     1921 aagactggaa cagctatttc tactgatttt tcctcgagaa gaccttgaca tgattttgaa
     1981 aatggattct ttacaggata taaaagcatt gttaacagga ttatttgtac aagataatgt
     2041 gaataaagat gccgtcacag atagattggc ttcagtggag actgatatgc ctctaacatt
     2101 gagacagcat agaataagtg cgacatcatc atcggaagag agtagtaaca aaggtcaaag
     2161 acagttgact gtatcgccgg tattgcaata cccagctttg actcatatgg ccatggaggc
     2221 cgaattcact ccgtccgagc tgcacaacga caacttctcg ctgtccacca tcgccgaggg
     2281 ctcccacccg aacagagaag cgcagcatag agccctctaa ggatccgtcg acctgcagcc
     2341 aagctaattc cgggcgaatt tcttatgatt tatgattttt attattaaat aagttataaa
     2401 aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt
     2461 cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagcatg aggtcgctct
     2521 tattgaccac acctctaccg gcatgcaagc ttggcgtaat catggtcata gctgtttcct
     2581 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     2641 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     2701 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     2761 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2821 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2881 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2941 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3001 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3061 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     3121 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     3181 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     3241 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     3301 ttatcgccac tggcagcagc cactagtaac aggattagca gagcgaggta tgtaggcggt
     3361 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     3421 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     3481 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3541 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3601 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3661 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3721 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3781 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3841 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3901 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3961 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     4021 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     4081 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     4141 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     4201 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     4261 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     4321 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     4381 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     4441 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     4501 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     4561 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     4621 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4681 ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac gaagcatctg tgcttcattt
     4741 tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat
     4801 ttttacagaa cagaaatgca acgcgaaagc gctattttac caacgaagaa tctgtgcttc
     4861 atttttgtaa aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc
     4921 tgcattttta cagaacagaa atgcaacgcg agagcgctat tttaccaaca aagaatctat
     4981 acttcttttt tgttctacaa aaatgcatcc cgagagcgct atttttctaa caaagcatct
     5041 tagattactt tttttctcct ttgtgcgctc tataatgcag tctcttgata actttttgca
     5101 ctgtaggtcc gttaaggtta gaagaaggct actttggtgt ctattttctc ttccataaaa
     5161 aaagcctgac tccacttccc gcgtttactg attactagcg aagctgcggg tgcatttttt
     5221 caagataaag gcatccccga ttatattcta taccgatgtg gattgcgcat actttgtgaa
     5281 cagaaagtga tagcgttgat gattcttcat tggtcagaaa attatgaacg gtttcttcta
     5341 ttttgtctct atatactacg tataggaaat gtttacattt tcgtattgtt ttcgattcac
     5401 tctatgaata gttcttacta caattttttt gtctaaagag taatactaga gataaacata
     5461 aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga tgggtaggtt
     5521 atatagggat atagcacaga gatatatagc aaagagatac ttttgagcaa tgtttgtgga
     5581 agcggtattc gcaatatttt agtagctcgt tacagtccgg tgcgtttttg gttttttgaa
     5641 agtgcgtctt cagagcgctt ttggttttca aaagcgctct gaagttccta tactttctag
     5701 agaataggaa cttcggaata ggaacttcaa agcgtttccg aaaacgagcg cttccgaaaa
     5761 tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc gcacctatat ctgcgtgttg
     5821 cctgtatata tatatacatg agaagaacgg catagtgcgt gtttatgctt aaatgcgtac
     5881 ttatatgcgt ctatttatgt aggatgaaag gtagtctagt acctcctgtg atattatccc
     5941 attccatgcg gggtatcgta tgcttccttc agcactaccc tttagctgtt ctatatgctg
     6001 ccactcctca attggattag tctcatcctt caatgctatc atttcctttg atattggatc
     6061 atactaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     6121 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     6181 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     6241 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     6301 gagagtgcac catagatcaa cgacattact atatatataa tataggaagc atttaataga
     6361 acagcatcgt aatatatgtg tactttgcag ttatgacgcc agatggcagt agtggaagat
     6421 attctttatt gaaaaatagc ttgtcacctt acgtacaatc ttgatccgga gcttttcttt
     6481 ttttgccgat taagaattaa ttcggtcgaa aaaagaaaag gagagggcca agagggaggg
     6541 cattggtgac tattgagcac gtgagtatac gtgattaagc acacaaaggc agcttggagt
     6601 atgtctgtta ttaatttcac aggtagttct ggtccattgg tgaaagtttg cggcttgcag
     6661 agcacagagg ccgcagaatg tgctctagat tccgatgctg acttgctggg tattatatgt
     6721 gtgcccaata gaaagagaac aattgacccg gttattgcaa ggaaaatttc aagtcttgta
     6781 aaagcatata aaaatagttc aggcactccg aaatacttgg ttggcgtgtt tcgtaatcaa
     6841 cctaaggagg atgttttggc tctggtcaat gattacggca ttgatatcgt ccaactgcat
     6901 ggagatgagt cgtggcaaga ataccaagag ttcctcggtt tgccagttat taaaagactc
     6961 gtatttccaa aagactgcaa catactactc agtgcagctt cacagaaacc tcattcgttt
     7021 attcccttgt ttgattcaga agcaggtggg acaggtgaac ttttggattg gaactcgatt
     7081 tctgactggg ttggaaggca agagagcccc gaaagcttac attttatgtt agctggtgga
     7141 ctgacgccag aaaatgttgg tgatgcgctt agattaaatg gcgttattgg tgttgatgta
     7201 agcggaggtg tggagacaaa tggtgtaaaa gactctaaca aaatagcaaa tttcgtcaaa
     7261 aatgctaaga aataggttat tactgagtag tatttattta agtattgttt gtgcacttgc
     7321 cgatctatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa
     7381 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     7441 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     7501 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     7561 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     7621 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     7681 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     7741 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     7801 ccgccgcgct taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg
     7861 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     7921 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     7981 gacggccagt gccaagcttt cgcgagctcg agatcccgag cttgcaaatt aaagccttcg
     8041 agcgtcccaa aaccttctca agcaaggttt tcagtataat gttacatgcg tacacgcgtc
     8101 tgtacagaaa aaaaagaaaa atttgaaata taaataacgt tcttaatact aacataacta
     8161 taaaaaaata aatagggacc tagacttcag gttgtctaac tccttccttt tcggttagag
     8221 cggatgtggg gggagggcgt gaatgtaagc gtgacataac taattacatg atatcgacaa
     8281 aggaaaaggg gcctgtttac tcacaggctt ttttcaagta ggtaattaag tcgtttctgt
     8341 ctttttcctt cttcaaccca ccaaaggcca tcttggtact tttttttttt tttttttttt
     8401 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
     8461 tttttttttt ttt
//
