LOCUS       sequence-387832-        8382 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GAL4 transcriptional activation domain (AD)-AP-2 
            sigma-2 fusion protein in yeast (yeast two-hybrid assays).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8382)
  AUTHORS   Mattera R, De Pace R, Bonifacino JS
  TITLE     The adaptor protein chaperone AAGAB stabilizes AP-4 complex 
            subunits.
  JOURNAL   Mol Biol Cell. 2022 Oct 1;33(12):ar109. doi: 
            10.1091/mbc.E22-05-0177. Epub 2022 Aug 17.
  PUBMED    35976721
REFERENCE   2  (bases 1 to 8382)
  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"; journalName: "Mol Biol 
            Cell."; date: "2022-10-1"; volume: "33(12):ar109. doi"; pages: " 
            10.1091/mbc.E22-05-0177. Epub 2022 Aug 17"
COMMENT     Sequence Label: pGADT7-AP-2 sigma-2
FEATURES             Location/Qualifiers
     source          1..8382
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        756..1460
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             1506..1526
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1542..1883
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="activation domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 activation domain
                     /translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     primer_bind     1889..1908
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1889..1907
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1914..1923
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1926..1952
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      2795..2982
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2850..2874)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(3099..4193)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIEATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(4194..4599)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4641..4657)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4641..4657)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4654..4676)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4665..4681
                     /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(4689..4695,4696..4713,4714..4719))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4734..4755
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    4810..4843
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4921..4954
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5023..5040)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5194..5782)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5274..5293)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5953..6744,6745..6813))
                     /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     6576..6595
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6814..6918)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6986..7004
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7042..7064)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7164..7183
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      7200..8364
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 cgagatccgg gatcgaagaa atgatggtaa atgaaatagg aaatcaagga gcatgaaggc
       61 aaaagacaaa tataagggtc gaacgaaaaa taaagtgaaa agtgttgata tgatgtattt
      121 ggctttgcgg cgccgaaaaa acgagtttac gcaattgcac aatcatgctg actctgtggc
      181 ggacccgcgc tcttgccggc ccggcgataa cgctgggcgt gaggctgtgc ccggcggagt
      241 tttttgcgcc tgcattttcc aaggtttacc ctgcgctaag gggcgagatt ggagaagcaa
      301 taagaatgcc ggttggggtt gcgatgatga cgaccacgac aactggtgtc attatttaag
      361 ttgccgaaag aacctgagtg catttgcaac atgagtatac tagaagaatg agccaagact
      421 tgcgagacgc gagtttgccg gtggtgcgaa caatagagcg accatgacct tgaaggtgag
      481 acgcgcataa ccgctagagt actttgaaga ggaaacagca atagggttgc taccagtata
      541 aatagacagg tacatacaac actggaaatg gttgtctgtt tgagtacgct ttcaattcat
      601 ttgggtgtgc actttattat gttacaatat ggaagggaac tttacacttc tcctatgcac
      661 atatattaat taaagtccaa tgctagtaga gaaggggggt aacacccctc cgcgctcttt
      721 tccgattttt ttctaaaccg tggaatattt cggatatcct tttgttgttt ccgggtgtac
      781 aatatggact tcctcttttc tggcaaccaa acccatacat cgggattcct ataatacctt
      841 cgttggtctc cctaacatgt aggtggcgga ggggagatat acaatagaac agataccaga
      901 caagacataa tgggctaaac aagactacac caattacact gcctcattga tggtggtaca
      961 taacgaacta atactgtagc cctagacttg atagccatca tcatatcgaa gtttcactac
     1021 cctttttcca tttgccatct attgaagtaa taataggcgc atgcaacttc ttttcttttt
     1081 ttttcttttc tctctccccc gttgttgtct caccatatcc gcaatgacaa aaaaatgatg
     1141 gaagacacta aaggaaaaaa ttaacgacaa agacagcacc aacagatgtc gttgttccag
     1201 agctgatgag gggtatctcg aagcacacga aactttttcc ttccttcatt cacgcacact
     1261 actctctaat gagcaacggt atacggcctt ccttccagtt acttgaattt gaaataaaaa
     1321 aaagtttgct gtcttgctat caagtataaa tagacctgca attattaatc ttttgtttcc
     1381 tcgtcattgt tctcgttccc tttcttcctt gtttcttttt ctgcacaata tttcaagcta
     1441 taccaagcat acaatcaact ccaagctttg caaagatgga taaagcggaa ttaattcccg
     1501 agcctccaaa aaagaagaga aaggtcgaat tgggtaccgc cgccaatttt aatcaaagtg
     1561 ggaatattgc tgatagctca ttgtccttca ctttcactaa cagtagcaac ggtccgaacc
     1621 tcataacaac tcaaacaaat tctcaagcgc tttcacaacc aattgcctcc tctaacgttc
     1681 atgataactt catgaataat gaaatcacgg ctagtaaaat tgatgatggt aataattcaa
     1741 aaccactgtc acctggttgg acggaccaaa ctgcgtataa cgcgtttgga atcactacag
     1801 ggatgtttaa taccactaca atggatgatg tatataacta tctattcgat gatgaagata
     1861 ccccaccaaa cccaaaaaaa gagatcttta atacgactca ctatagggcg agcgccgcca
     1921 tggagtaccc atacgacgta ccagattacg ctcatatggc catggaggcc agtgaattca
     1981 tgatccgctt tatcctcatc cagaaccggg caggcaagac acgcctggcc aagtggtata
     2041 tgcagttcga tgatgacgag aagcagaagt tgatcgagga agtgcacgcc gtggtcaccg
     2101 tcggggatgc caagcacacc aactttgtgg agttccggaa cttcaagatc atctaccggc
     2161 gctacgctgg cctctacttc tgcatctgcg ttgacgtcaa cgacaacaat ctggcctatc
     2221 ttgaggccat ccacaacttc gtagaagtgt taaatgaata cttccacaat gtctgtgaac
     2281 tggacctggt gttcaacttc tacaaggttt acacggtggt agatgagatg ttcctggcag
     2341 gagagatccg agagacgagc cagacgaagg tgctgaagca gctgctgatg ctacagtccc
     2401 tggagtgact cgagctgcag atgaatcgta gatactgaaa aaccccgcaa gttcacttca
     2461 actgtgcatc gtgcaccatc tcaatttctt tcatttatac atcgttttgc cttcttttat
     2521 gtaactatac tcctctaagt ttcaatcttg gccatgtaac ctctgatcta tagaattttt
     2581 taaatgacta gaattaatgc ccatcttttt tttggaccta aattcttcat gaaaatatat
     2641 tacgagggct tattcagaag ctttggactt cttcgccaga ggtttggtca agtctccaat
     2701 caaggttgtc ggcttgtcta ccttgccaga aatttacgaa aagatggaaa agggtcaaat
     2761 cgttggtaga tacgttgttg acacttctaa ataagcgaat ttcttatgat ttatgatttt
     2821 tattattaaa taagttataa aaaaaataag tgtatacaaa ttttaaagtg actcttaggt
     2881 tttaaaacga aaattcttat tcttgagtaa ctctttcctg taggtcaggt tgctttctca
     2941 ggtatagcat gaggtcgctc ttattgacca cacctctacc ggccggtcga aattccccta
     3001 ccctatgaac atattccatt ttgtaatttc gtgtcgtttc tattatgaat ttcatttata
     3061 aagtttatgt acaaatatca taaaaaaaga gaatcttttt aagcaaggat tttcttaact
     3121 tcttcggcga cagcatcacc gacttcggtg gtactgttgg aaccacctaa atcaccagtt
     3181 ctgatacctg catccaaaac ctttttaact gcatcttcaa tggccttacc ttcttcaggc
     3241 aagttcaatg acaatttcaa catcattgca gcagacaaga tagtggcgat agggttgacc
     3301 ttattctttg gcaaatctgg agcagaaccg tggcatggtt cgtacaaacc aaatgcggtg
     3361 ttcttgtctg gcaaagaggc caaggacgca gatggcaaca aacccaagga acctgggata
     3421 acggaggctt catcggagat gatatcacca aacatgttgc tggtgattat aataccattt
     3481 aggtgggttg ggttcttaac taggatcatg gcggcagaat caatcaattg atgttgaacc
     3541 ttcaatgtag gaaattcgtt cttgatggtt tcctccacag tttttctcca taatcttgaa
     3601 gaggccaaaa cattagcttt atccaaggac caaataggca atggtggctc atgttgtagg
     3661 gccatgaaag cggccattct tgtgattctt tgcacttctg gaacggtgta ttgttcacta
     3721 tcccaagcga caccatcacc atcgtcttcc tttctcttac caaagtaaat acctcccact
     3781 aattctctga caacaacgaa gtcagtacct ttagcaaatt gtggcttgat tggagataag
     3841 tctaaaagag agtcggatgc aaagttacat ggtcttaagt tggcgtacaa ttgaagttct
     3901 ttacggattt ttagtaaacc ttgttcaggt ctaacactac ctgtacccca tttaggacca
     3961 cccacagcac ctaacaaaac ggcatcagcc ttcttggagg cttccagcgc ctcatctgga
     4021 agtgggacac ctgtagcttc gatagcagca ccaccaatta aatgattttc gaaatcgaac
     4081 ttgacattgg aacgaacatc agaaatagct ttaagaacct taatggcttc ggctgtgatt
     4141 tcttgaccaa cgtggtcacc tggcaaaacg acgatcttct taggggcaga cattagaatg
     4201 gtatatcctt gaaatatata tatatattgc tgaaatgtaa aaggtaagaa aagttagaaa
     4261 gtaagacgat tgctaaccac ctattggaaa aaacaatagg tccttaaata atattgtcaa
     4321 cttcaagtat tgtgatgcaa gcatttagtc atgaacgctt ctctattcta tatgaaaagc
     4381 cggttccggc gctctcacct ttcctttttc tcccaatttt tcagttgaaa aaggtatatg
     4441 cgtcaggcga cctctgaaat taacaaaaaa tttccagtca tcgaatttga ttctgtgcga
     4501 tagcgcccct gtgtgttctc gttatgttga ggaaaaaaat aatggttgct aagagattcg
     4561 aactcttgca tcttacgata cctgagtatt cccacagttg ggggatctcg actctagcta
     4621 gaggatcaat tcgtaatcat gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4681 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4741 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4801 tgccagctga taacttcgta taatgtatgc tatacgaagt tattaggtct gaagaggagt
     4861 ttacgtccag ccaagctagc ttggctgcag gtcgagcggc cgcgatccgg aacccttaat
     4921 ataacttcgt ataatgtatg ctatacgaag ttatcagctg cattaatgaa tcggccaacg
     4981 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5041 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5101 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5161 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5221 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5281 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5341 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5401 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5461 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5521 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5581 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5641 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5701 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5761 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5821 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5881 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5941 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6001 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6061 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6121 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6181 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6241 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6301 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6361 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6421 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6481 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6541 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6601 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6661 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6721 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6781 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6841 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6901 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     6961 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     7021 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     7081 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     7141 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccataa
     7201 cgcatttaag cataaacacg cactatgccg ttcttctcat gtatatatat atacaggcaa
     7261 cacgcagata taggtgcgac gtgaacagtg agctgtatgt gcgcagctcg cgttgcattt
     7321 tcggaagcgc tcgttttcgg aaacgctttg aagttcctat tccgaagttc ctattctcta
     7381 gctagaaagt ataggaactt cagagcgctt ttgaaaacca aaagcgctct gaagacgcac
     7441 tttcaaaaaa ccaaaaacgc accggactgt aacgagctac taaaatattg cgaataccgc
     7501 ttccacaaac attgctcaaa agtatctctt tgctatatat ctctgtgcta tatccctata
     7561 taacctaccc atccaccttt cgctccttga acttgcatct aaactcgacc tctacatttt
     7621 ttatgtttat ctctagtatt actctttaga caaaaaaatt gtagtaagaa ctattcatag
     7681 agtgaatcga aaacaatacg aaaatgtaaa catttcctat acgtagtata tagagacaaa
     7741 atagaagaaa ccgttcataa ttttctgacc aatgaagaat catcaacgct atcactttct
     7801 gttcacaaag tatgcgcaat ccacatcggt atagaatata atcggggatg cctttatctt
     7861 gaaaaaatgc acccgcagct tcgctagtaa tcagtaaacg cgggaagtgg agtcaggctt
     7921 tttttatgga agagaaaata gacaccaaag tagccttctt ctaaccttaa cggacctaca
     7981 gtgcaaaaag ttatcaagag actgcattat agagcgcaca aaggagaaaa aaagtaatct
     8041 aagatgcttt gttagaaaaa tagcgctctc gggatgcatt tttgtagaac aaaaaagaag
     8101 tatagattct ttgttggtaa aatagcgctc tcgcgttgca tttctgttct gtaaaaatgc
     8161 agctcagatt ctttgtttga aaaattagcg ctctcgcgtt gcatttttgt tttacaaaaa
     8221 tgaagcacag attcttcgtt ggtaaaatag cgctttcgcg ttgcatttct gttctgtaaa
     8281 aatgcagctc agattctttg tttgaaaaat tagcgctctc gcgttgcatt tttgttctac
     8341 aaaatgaagc acagatgctt cgttgcttgc atgcctgcag gt
//
