LOCUS       sequence-177490-        8696 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 8696)
  TITLE     Rathjen lab plasmids
REFERENCE   2  (bases 1 to 8696)
  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-mCALEBsplice.cyt (aa 465-566)
FEATURES             Location/Qualifiers
     source          1..8696
                     /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      2577..2764
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2632..2656)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2788..2804)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2788..2804)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2801..2823)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2812..2828
                     /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(2836..2842,2843..2860,2861..2866))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2881..2902
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3019..3036)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3190..3778)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3270..3289)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3949..4740,4741..4809))
                     /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     4572..4591
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4810..4914)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4941..6283
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5904..5951)
                     /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     6324..6342
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6380..6402)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6502..6521
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6542..6823
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             6824..7498
                     /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(7601..8056)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7738..7759)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7950..7969
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8182..8204
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8196..8213
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8197..8213
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      complement(8247..8494)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     8460..8478
                     /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 aaatgtaagg aaattttgcg acactccccg tgtctcctcc ccccatgccc
     2341 gtgccttggc tcactatgat aacattgtct gtcaggacga ccccagcgct ccccacaaaa
     2401 tccaggaccc tctcaagtcc cgcctgaagg aggaagagtc ctttaacatc cagaactcca
     2461 tgtcacccaa acttgagggt ggcaaaggtg accaggatga cttgggggtg aactgtctgc
     2521 agaataacct aacctgagac tgaggatccg tcgacctgca gccaagctaa ttccgggcga
     2581 atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata agtgtataca
     2641 aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt aactctttcc
     2701 tgtaggtcag gttgctttct caggtatagc atgaggtcgc tcttattgac cacacctcta
     2761 ccggcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     2821 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     2881 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2941 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3001 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3061 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3121 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3181 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3241 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3301 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3361 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3421 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3481 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3541 agccactagt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3601 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3661 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3721 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3781 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3841 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3901 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3961 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4021 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4081 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4141 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4201 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4261 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4321 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4381 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4441 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4501 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4561 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4621 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4681 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4741 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4801 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4861 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4921 tccccgaaaa gtgccacctg aacgaagcat ctgtgcttca ttttgtagaa caaaaatgca
     4981 acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca gaacagaaat
     5041 gcaacgcgaa agcgctattt taccaacgaa gaatctgtgc ttcatttttg taaaacaaaa
     5101 atgcaacgcg agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca
     5161 gaaatgcaac gcgagagcgc tattttacca acaaagaatc tatacttctt ttttgttcta
     5221 caaaaatgca tcccgagagc gctatttttc taacaaagca tcttagatta ctttttttct
     5281 cctttgtgcg ctctataatg cagtctcttg ataacttttt gcactgtagg tccgttaagg
     5341 ttagaagaag gctactttgg tgtctatttt ctcttccata aaaaaagcct gactccactt
     5401 cccgcgttta ctgattacta gcgaagctgc gggtgcattt tttcaagata aaggcatccc
     5461 cgattatatt ctataccgat gtggattgcg catactttgt gaacagaaag tgatagcgtt
     5521 gatgattctt cattggtcag aaaattatga acggtttctt ctattttgtc tctatatact
     5581 acgtatagga aatgtttaca ttttcgtatt gttttcgatt cactctatga atagttctta
     5641 ctacaatttt tttgtctaaa gagtaatact agagataaac ataaaaaatg tagaggtcga
     5701 gtttagatgc aagttcaagg agcgaaaggt ggatgggtag gttatatagg gatatagcac
     5761 agagatatat agcaaagaga tacttttgag caatgtttgt ggaagcggta ttcgcaatat
     5821 tttagtagct cgttacagtc cggtgcgttt ttggtttttt gaaagtgcgt cttcagagcg
     5881 cttttggttt tcaaaagcgc tctgaagttc ctatactttc tagagaatag gaacttcgga
     5941 ataggaactt caaagcgttt ccgaaaacga gcgcttccga aaatgcaacg cgagctgcgc
     6001 acatacagct cactgttcac gtcgcaccta tatctgcgtg ttgcctgtat atatatatac
     6061 atgagaagaa cggcatagtg cgtgtttatg cttaaatgcg tacttatatg cgtctattta
     6121 tgtaggatga aaggtagtct agtacctcct gtgatattat cccattccat gcggggtatc
     6181 gtatgcttcc ttcagcacta ccctttagct gttctatatg ctgccactcc tcaattggat
     6241 tagtctcatc cttcaatgct atcatttcct ttgatattgg atcatactaa gaaaccatta
     6301 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     6361 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     6421 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     6481 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatagat
     6541 caacgacatt actatatata taatatagga agcatttaat agaacagcat cgtaatatat
     6601 gtgtactttg cagttatgac gccagatggc agtagtggaa gatattcttt attgaaaaat
     6661 agcttgtcac cttacgtaca atcttgatcc ggagcttttc tttttttgcc gattaagaat
     6721 taattcggtc gaaaaaagaa aaggagaggg ccaagaggga gggcattggt gactattgag
     6781 cacgtgagta tacgtgatta agcacacaaa ggcagcttgg agtatgtctg ttattaattt
     6841 cacaggtagt tctggtccat tggtgaaagt ttgcggcttg cagagcacag aggccgcaga
     6901 atgtgctcta gattccgatg ctgacttgct gggtattata tgtgtgccca atagaaagag
     6961 aacaattgac ccggttattg caaggaaaat ttcaagtctt gtaaaagcat ataaaaatag
     7021 ttcaggcact ccgaaatact tggttggcgt gtttcgtaat caacctaagg aggatgtttt
     7081 ggctctggtc aatgattacg gcattgatat cgtccaactg catggagatg agtcgtggca
     7141 agaataccaa gagttcctcg gtttgccagt tattaaaaga ctcgtatttc caaaagactg
     7201 caacatacta ctcagtgcag cttcacagaa acctcattcg tttattccct tgtttgattc
     7261 agaagcaggt gggacaggtg aacttttgga ttggaactcg atttctgact gggttggaag
     7321 gcaagagagc cccgaaagct tacattttat gttagctggt ggactgacgc cagaaaatgt
     7381 tggtgatgcg cttagattaa atggcgttat tggtgttgat gtaagcggag gtgtggagac
     7441 aaatggtgta aaagactcta acaaaatagc aaatttcgtc aaaaatgcta agaaataggt
     7501 tattactgag tagtatttat ttaagtattg tttgtgcact tgccgatcta tgcggtgtga
     7561 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaatatt
     7621 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     7681 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     7741 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     7801 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     7861 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     7921 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     7981 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     8041 ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     8101 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     8161 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgccaagc
     8221 tttcgcgagc tcgagatccc gagcttgcaa attaaagcct tcgagcgtcc caaaaccttc
     8281 tcaagcaagg ttttcagtat aatgttacat gcgtacacgc gtctgtacag aaaaaaaaga
     8341 aaaatttgaa atataaataa cgttcttaat actaacataa ctataaaaaa ataaataggg
     8401 acctagactt caggttgtct aactccttcc ttttcggtta gagcggatgt ggggggaggg
     8461 cgtgaatgta agcgtgacat aactaattac atgatatcga caaaggaaaa ggggcctgtt
     8521 tactcacagg cttttttcaa gtaggtaatt aagtcgtttc tgtctttttc cttcttcaac
     8581 ccaccaaagg ccatcttggt actttttttt tttttttttt tttttttttt tttttttttt
     8641 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttt
//
