LOCUS       sequence-376859-        5239 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express GST-tagged NEDD4L WW34 tandem domains in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5239)
  AUTHORS   Wright KM, Nathan S, Jiang H, Xia W, Kim H, Chakouri N, Nwafor JN, 
            Fossier L, Srinivasan L, Chen Z, Boronina T, Post J, Paul S, Cole 
            RN, Ben-Johny M, Cole PA, Gabelli SB
  TITLE     NEDD4L intramolecular interactions regulate its auto and substrate 
            Na(V)1.5 ubiquitination.
  JOURNAL   J Biol Chem. 2024 Mar;300(3):105715. doi: 10.1016/j.jbc.2024.105715.
            Epub 2024 Feb 2.
  PUBMED    38309503
REFERENCE   2  (bases 1 to 5239)
  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: "J Biol 
            Chem."; date: "2024-03"; volume: "300(3):105715. doi"; pages: " 
            10.1016/j.jbc.2024.105715. Epub 2024 Feb 2"
COMMENT     Sequence Label: pGEX6P1-NEDD4L_WW34
FEATURES             Location/Qualifiers
     source          1..5239
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2..529
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(22..39)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(22..38)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(31..53)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(631..636,637..652,653..659)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    667..683
                     /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             706..1359
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1317..1339
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1366..1389
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1737..1759)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1904..1922)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1990..2094
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2095..2163,2164..2955)
                     /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(2313..2332)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3126..3714
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3615..3634
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3868..3885
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3958..4035
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4036..5118
                     /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(4055..4074)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5167..5172,5173..5190,5191..5197)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5205..5221
                     /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     join(5229..5239,1..6)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5229..5239,1..6)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tatgaccatg attacggatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
       61 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
      121 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
      181 ctttgcctgg tttccggcac cagaagcggt gccggaaagc tggctggagt gcgatcttcc
      241 tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat
      301 ctacaccaac gtgacctatc ccattacggt caatccgccg tttgttccca cggagaatcc
      361 gacgggttgt tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac
      421 gcgaattatt tttgatggcg ttggaattag cttatcgact gcacggtgca ccaatgcttc
      481 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata
      541 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa
      601 cggttctggc aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt
      661 gtggaattgt gagcggataa caatttcaca caggaaacag tattcatgtc ccctatacta
      721 ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga atatcttgaa
      781 gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg aaacaaaaag
      841 tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga tgttaaatta
      901 acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt gggtggttgt
      961 ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat tagatacggt
     1021 gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt tcttagcaag
     1081 ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata tttaaatggt
     1141 gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt tttatacatg
     1201 gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg tattgaagct
     1261 atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc tttgcagggc
     1321 tggcaagcca cgtttggtgg tggcgaccat cctccaaaat cggatctgga agttctgttc
     1381 caggggcccc tgggatccca caaagtcaca cagagcttct tgccacccgg ctgggaaatg
     1441 aggatagcgc caaacggccg gcccttcttc attgatcata acacaaagac tacaacctgg
     1501 gaagatccac gtttgaaatt tccagtacat atgcggtcaa agacatcttt aaaccccaat
     1561 gaccttggcc cccttcctcc tggctgggaa gaaagaattc acttggatgg ccgaacgttt
     1621 tatattgatc ataatagcaa aattactcag tgggaagacc caagactgta gctcgagcgg
     1681 ccgcatcgtg actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc
     1741 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     1801 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag
     1861 tcacgtagcg atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta
     1921 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     1981 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2041 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     2101 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     2161 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     2221 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     2281 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     2341 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     2401 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     2461 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     2521 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     2581 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     2641 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     2701 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     2761 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     2821 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     2881 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     2941 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     3001 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3061 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3121 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3181 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3241 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3301 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3361 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3421 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3481 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3541 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3601 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3661 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3721 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3781 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3841 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3901 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac
     3961 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
     4021 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
     4081 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
     4141 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
     4201 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
     4261 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
     4321 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
     4381 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
     4441 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
     4501 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
     4561 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt
     4621 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa
     4681 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag
     4741 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc
     4801 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat
     4861 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg
     4921 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     4981 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca cgctagcgcc caatacgcaa
     5041 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     5101 ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc
     5161 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     5221 atttcacaca ggaaacagc
//
