LOCUS       sequence-201230-        5134 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of RKK mutant WW domain 1 of YAP; RRK mutant 
            binds to PPxY where Y is phosphorylated.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5134)
  TITLE     WW domains of YAP as a wild type and its RRK mutant that binds to 
            PPx(phospho)Y peptides
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5134)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pGEX2TK-YAP-WW1-RRK
FEATURES             Location/Qualifiers
     source          1..5134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(47..52,53..68,69..75)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    83..99
                     /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)."
     CDS             122..775
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     733..755
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             782..799
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1048..1070)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1215..1233)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1301..1405
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1406..1474,1475..2266)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1624..1643)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2437..3025
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2926..2945
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3179..3196
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3269..3346
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3347..4429
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3366..3385)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4478..4483,4484..4501,4502..4508)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4516..4532
                     /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)."
     primer_bind     4540..4556
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4540..4556
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4552..5079
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4572..4589)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4572..4588)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4581..4603)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tgcgccgaca tcataacggt tctggcaaat attctgaaat gagctgttga caattaatca
       61 tcggctcgta taatgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagtatt
      121 catgtcccct atactaggtt attggaaaat taagggcctt gtgcaaccca ctcgacttct
      181 tttggaatat cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg aaggtgataa
      241 atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttcctt attatattga
      301 tggtgatgtt aaattaacac agtctatggc catcatacgt tatatagctg acaagcacaa
      361 catgttgggt ggttgtccaa aagagcgtgc agagatttca atgcttgaag gagcggtttt
      421 ggatattaga tacggtgttt cgagaattgc atatagtaaa gactttgaaa ctctcaaagt
      481 tgattttctt agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa
      541 aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatg acgctcttga
      601 tgttgtttta tacatggacc caatgtgcct ggatgcgttc ccaaaattag tttgttttaa
      661 aaaacgtatt gaagctatcc cacaaattga taagtacttg aaatccagca agtatatagc
      721 atggcctttg cagggctggc aagccacgtt tggtggtggc gaccatcctc caaaatcgga
      781 tctggttccg cgtggatctc gtcgtgcatc tgttggatcc cagtcttctt ttgagatacc
      841 tgatgatgta cctctgccag caggttggga gatggcaaag acatcttctg gtcagagata
      901 cttcagaaat agaatcgata aaacaacaac atggcaggac cccaggaagg ccatgctgtc
      961 ccagatgaac gtcacagccc ccaccagtcg aattcatcgt gactgactga cgatctgcct
     1021 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     1081 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     1141 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtataattc
     1201 ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     1261 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1321 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1381 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     1441 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     1501 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     1561 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     1621 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     1681 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     1741 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     1801 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     1861 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     1921 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     1981 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2041 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2101 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     2161 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2221 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2281 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2341 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2401 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2461 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2521 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2581 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2641 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2701 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2761 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     2821 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     2881 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     2941 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3001 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3061 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3121 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3181 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3241 gtgcggtatt tcacaccgca taaattccga caccatcgaa tggcgcaaaa cctttcgcgg
     3301 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
     3361 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     3421 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     3481 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     3541 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     3601 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     3661 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     3721 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     3781 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     3841 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     3901 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     3961 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     4021 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     4081 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     4141 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     4201 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     4261 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     4321 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4381 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     4441 ttaatgtaag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     4501 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     4561 attacggatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     4621 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     4681 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg ctttgcctgg
     4741 tttccggcac cagaagcggt gccggaaagc tggctggagt gcgatcttcc tgaggccgat
     4801 actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat ctacaccaac
     4861 gtgacctatc ccattacggt caatccgccg tttgttccca cggagaatcc gacgggttgt
     4921 tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac gcgaattatt
     4981 tttgatggcg ttggaattag cttatcgact gcacggtgca ccaatgcttc tggcgtcagg
     5041 cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg
     5101 ctcaaggcgc actcccgttc tggataatgt tttt
//
