LOCUS       sequence-398153-        5315 bp    DNA     circular SYN 15-APR-2025
DEFINITION  N-terminal GST fusion of RNF146(100-182) WWE domain with a TEV 
            protease site located between the GST tag and Af1521 and a myc tag 
            on the C terminus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5315)
  TITLE     WWE domain and PAR dynamics
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5315)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pGEX-4T-3-GST-Tev-RNF146(100-182)-myc
FEATURES             Location/Qualifiers
     source          1..5315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(182..187,188..203,204..210)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    218..234
                     /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             257..910
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     868..890
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             917..934
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             953..973
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1271..1300
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1364..1386)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1531..1549)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1617..1721
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1722..1790,1791..2582)
                     /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(1940..1959)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2753..3341
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3242..3261
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3495..3512
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3585..3662
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3663..4745
                     /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(3682..3701)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4794..4799,4800..4817,4818..4824)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4832..4848
                     /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     4856..4872
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4856..4872
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4868..5315,1..80)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4888..4905)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4888..4904)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4897..4919)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gcttatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
       61 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
      121 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
      181 gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac
      241 acaggaaaca gtattcatgt cccctatact aggttattgg aaaattaagg gccttgtgca
      301 acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg
      361 cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct
      421 tccttattat attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat
      481 agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct
      541 tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt
      601 tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga
      661 tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt
      721 gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa
      781 attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc
      841 cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca
      901 tcctccaaaa tcggatctgg ttccgcgtgg atccccgaat tcccgggtcg acgaaaatct
      961 atattttcaa ggtggaaacg gtgaatatgc gtggtattac gagggtcgta atggttggtg
     1021 gcagtacgac gaacgtacga gccgtgaact ggaggacgca tttagcaaag gtaagaagaa
     1081 caccgaaatg ctgattgctg gtttcctgta tgtggccgat ttggagaaca tggtccagta
     1141 ccgccgcaac gagcacggcc gtagacgcaa gattaaacgt gatattatcg atatcccgaa
     1201 aaagggtgtt gcgggtctcc gtagcggtgg cggcggctct ggtggcggcg gctccggtgg
     1261 tggcggctcg gagcaaaaac tgatcagcga ggaagacttg taagcggccg catcgtgact
     1321 gactgacgat ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     1381 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     1441 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     1501 gcggagtgta taattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
     1561 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     1621 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1681 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1741 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1801 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1861 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1921 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     1981 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2041 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2101 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2161 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2221 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     2281 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2341 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2401 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2461 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2521 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2581 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2641 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2701 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2761 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2821 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2881 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2941 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3001 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3061 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3121 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3181 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3241 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3301 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3361 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3421 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3481 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3541 ttctccttac gcatctgtgc ggtatttcac accgcataaa ttccgacacc atcgaatggc
     3601 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     3661 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     3721 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     3781 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     3841 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     3901 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     3961 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     4021 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     4081 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     4141 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     4201 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     4261 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     4321 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     4381 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     4441 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     4501 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     4561 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     4621 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     4681 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     4741 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacccca ggctttacac
     4801 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     4861 aacagctatg accatgatta cggattcact ggccgtcgtt ttacaacgtc gtgactggga
     4921 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     4981 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     5041 atggcgcttt gcctggtttc cggcaccaga agcggtgccg gaaagctggc tggagtgcga
     5101 tcttcctgag gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg gttacgatgc
     5161 gcccatctac accaacgtga cctatcccat tacggtcaat ccgccgtttg ttcccacgga
     5221 gaatccgacg ggttgttact cgctcacatt taatgttgat gaaagctggc tacaggaagg
     5281 ccagacgcga attatttttg atggcgttgg aatta
//
