LOCUS       sequence-177487-        8603 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 8603)
  TITLE     Rathjen lab plasmids
REFERENCE   2  (bases 1 to 8603)
  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-mCALEBlong.cyt (aa 522-594)
FEATURES             Location/Qualifiers
     source          1..8603
                     /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      2484..2671
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2539..2563)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2695..2711)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2695..2711)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2708..2730)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2719..2735
                     /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(2743..2749,2750..2767,2768..2773))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2788..2809
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2926..2943)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3097..3685)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3177..3196)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3856..4647,4648..4716))
                     /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     4479..4498
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4717..4821)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4848..6190
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5811..5858)
                     /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     6231..6249
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6287..6309)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6409..6428
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6449..6730
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             6731..7405
                     /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(7508..7963)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7645..7666)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7857..7876
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8089..8111
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8103..8120
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8104..8120
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      complement(8154..8401)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     8367..8385
                     /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 tccgtccgag ctgcacaacg acaacttctc gctgtccacc atcgccgagg
     2281 gctcccaccc gaacgatgac cctggcgcac cccacaagct gcaggacccg ctgaagcctg
     2341 gcctgaagga cgaggaaccg ctcagcatcc tcagcaccgc gccagaggag ggcagcaagg
     2401 gcgaaccagg gggctgcggg gtgccctgcc tgcacaacaa cctggggtag ggatccgtcg
     2461 acctgcagcc aagctaattc cgggcgaatt tcttatgatt tatgattttt attattaaat
     2521 aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa
     2581 aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagcatg
     2641 aggtcgctct tattgaccac acctctaccg gcatgcaagc ttggcgtaat catggtcata
     2701 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     2761 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     2821 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     2881 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     2941 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3001 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3061 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3121 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3181 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3241 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3301 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3361 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3421 aagacacgac ttatcgccac tggcagcagc cactagtaac aggattagca gagcgaggta
     3481 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3541 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3601 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3661 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3721 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3781 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3841 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3901 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3961 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     4021 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4081 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4141 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4201 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4261 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4321 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4381 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4441 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4501 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4561 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4621 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4681 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4741 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4801 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac gaagcatctg
     4861 tgcttcattt tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc
     4921 tgagctgcat ttttacagaa cagaaatgca acgcgaaagc gctattttac caacgaagaa
     4981 tctgtgcttc atttttgtaa aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa
     5041 gaatctgagc tgcattttta cagaacagaa atgcaacgcg agagcgctat tttaccaaca
     5101 aagaatctat acttcttttt tgttctacaa aaatgcatcc cgagagcgct atttttctaa
     5161 caaagcatct tagattactt tttttctcct ttgtgcgctc tataatgcag tctcttgata
     5221 actttttgca ctgtaggtcc gttaaggtta gaagaaggct actttggtgt ctattttctc
     5281 ttccataaaa aaagcctgac tccacttccc gcgtttactg attactagcg aagctgcggg
     5341 tgcatttttt caagataaag gcatccccga ttatattcta taccgatgtg gattgcgcat
     5401 actttgtgaa cagaaagtga tagcgttgat gattcttcat tggtcagaaa attatgaacg
     5461 gtttcttcta ttttgtctct atatactacg tataggaaat gtttacattt tcgtattgtt
     5521 ttcgattcac tctatgaata gttcttacta caattttttt gtctaaagag taatactaga
     5581 gataaacata aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga
     5641 tgggtaggtt atatagggat atagcacaga gatatatagc aaagagatac ttttgagcaa
     5701 tgtttgtgga agcggtattc gcaatatttt agtagctcgt tacagtccgg tgcgtttttg
     5761 gttttttgaa agtgcgtctt cagagcgctt ttggttttca aaagcgctct gaagttccta
     5821 tactttctag agaataggaa cttcggaata ggaacttcaa agcgtttccg aaaacgagcg
     5881 cttccgaaaa tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc gcacctatat
     5941 ctgcgtgttg cctgtatata tatatacatg agaagaacgg catagtgcgt gtttatgctt
     6001 aaatgcgtac ttatatgcgt ctatttatgt aggatgaaag gtagtctagt acctcctgtg
     6061 atattatccc attccatgcg gggtatcgta tgcttccttc agcactaccc tttagctgtt
     6121 ctatatgctg ccactcctca attggattag tctcatcctt caatgctatc atttcctttg
     6181 atattggatc atactaagaa accattatta tcatgacatt aacctataaa aataggcgta
     6241 tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     6301 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     6361 agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc
     6421 agattgtact gagagtgcac catagatcaa cgacattact atatatataa tataggaagc
     6481 atttaataga acagcatcgt aatatatgtg tactttgcag ttatgacgcc agatggcagt
     6541 agtggaagat attctttatt gaaaaatagc ttgtcacctt acgtacaatc ttgatccgga
     6601 gcttttcttt ttttgccgat taagaattaa ttcggtcgaa aaaagaaaag gagagggcca
     6661 agagggaggg cattggtgac tattgagcac gtgagtatac gtgattaagc acacaaaggc
     6721 agcttggagt atgtctgtta ttaatttcac aggtagttct ggtccattgg tgaaagtttg
     6781 cggcttgcag agcacagagg ccgcagaatg tgctctagat tccgatgctg acttgctggg
     6841 tattatatgt gtgcccaata gaaagagaac aattgacccg gttattgcaa ggaaaatttc
     6901 aagtcttgta aaagcatata aaaatagttc aggcactccg aaatacttgg ttggcgtgtt
     6961 tcgtaatcaa cctaaggagg atgttttggc tctggtcaat gattacggca ttgatatcgt
     7021 ccaactgcat ggagatgagt cgtggcaaga ataccaagag ttcctcggtt tgccagttat
     7081 taaaagactc gtatttccaa aagactgcaa catactactc agtgcagctt cacagaaacc
     7141 tcattcgttt attcccttgt ttgattcaga agcaggtggg acaggtgaac ttttggattg
     7201 gaactcgatt tctgactggg ttggaaggca agagagcccc gaaagcttac attttatgtt
     7261 agctggtgga ctgacgccag aaaatgttgg tgatgcgctt agattaaatg gcgttattgg
     7321 tgttgatgta agcggaggtg tggagacaaa tggtgtaaaa gactctaaca aaatagcaaa
     7381 tttcgtcaaa aatgctaaga aataggttat tactgagtag tatttattta agtattgttt
     7441 gtgcacttgc cgatctatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     7501 catcaggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     7561 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     7621 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     7681 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     7741 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     7801 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     7861 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     7921 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtccattcg ccattcaggc
     7981 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     8041 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
     8101 gttgtaaaac gacggccagt gccaagcttt cgcgagctcg agatcccgag cttgcaaatt
     8161 aaagccttcg agcgtcccaa aaccttctca agcaaggttt tcagtataat gttacatgcg
     8221 tacacgcgtc tgtacagaaa aaaaagaaaa atttgaaata taaataacgt tcttaatact
     8281 aacataacta taaaaaaata aatagggacc tagacttcag gttgtctaac tccttccttt
     8341 tcggttagag cggatgtggg gggagggcgt gaatgtaagc gtgacataac taattacatg
     8401 atatcgacaa aggaaaaggg gcctgtttac tcacaggctt ttttcaagta ggtaattaag
     8461 tcgtttctgt ctttttcctt cttcaaccca ccaaaggcca tcttggtact tttttttttt
     8521 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
     8581 tttttttttt tttttttttt ttt
//
