LOCUS       sequence-372418-        7901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express the catalytically inactive GST-tagged NEDD4L FL in bacterial
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7901)
  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 7901)
  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_FL-CS
FEATURES             Location/Qualifiers
     source          1..7901
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(142..147,148..163,164..170)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    178..194
                     /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             217..870
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     828..850
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             877..900
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(3910..3932)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4077..4095)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4163..4267
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4268..4336,4337..5128)
                     /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(4486..4505)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5299..5887
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5788..5807
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6041..6058
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6131..6208
                     /gene="lacI"
                     /label=lacI promoter
     CDS             6209..7291
                     /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(6228..6247)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(7340..7345,7346..7363,7364..7370)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7378..7394
                     /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     7402..7418
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7402..7418
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(7414..7901,1..40)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(7434..7451)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7434..7450)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7443..7465)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc
       61 gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg
      121 caaatattct gaaatgagct gttgacaatt aatcatcggc tcgtataatg tgtggaattg
      181 tgagcggata acaatttcac acaggaaaca gtattcatgt cccctatact aggttattgg
      241 aaaattaagg gccttgtgca acccactcga cttcttttgg aatatcttga agaaaaatat
      301 gaagagcatt tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg
      361 ggtttggagt ttcccaatct tccttattat attgatggtg atgttaaatt aacacagtct
      421 atggccatca tacgttatat agctgacaag cacaacatgt tgggtggttg tccaaaagag
      481 cgtgcagaga tttcaatgct tgaaggagcg gttttggata ttagatacgg tgtttcgaga
      541 attgcatata gtaaagactt tgaaactctc aaagttgatt ttcttagcaa gctacctgaa
      601 atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat atttaaatgg tgatcatgta
      661 acccatcctg acttcatgtt gtatgacgct cttgatgttg ttttatacat ggacccaatg
      721 tgcctggatg cgttcccaaa attagtttgt tttaaaaaac gtattgaagc tatcccacaa
      781 attgataagt acttgaaatc cagcaagtat atagcatggc ctttgcaggg ctggcaagcc
      841 acgtttggtg gtggcgacca tcctccaaaa tcggatctgg aagttctgtt ccaggggccc
      901 ctgggagtac gcgccggctc catggcgacc gggctcgggg agccggtcta tggactttcc
      961 gaagacgagg gagagtcccg tattctcaga gtaaaagttg tttctggaat tgatctcgcc
     1021 aaaaaggaca tctttggagc cagtgatccg tatgtgaaac tttcattgta cgtagcggat
     1081 gagaatagag aacttgcttt ggtccagaca aaaacaatta aaaagacact gaacccaaaa
     1141 tggaatgaag aattttattt cagggtaaac ccatctaatc acagactcct atttgaagta
     1201 tttgacgaaa atagactgac acgagacgac ttcctgggcc aggtggacgt gccccttagt
     1261 caccttccga cagaagatcc aaccatggag cgaccctata catttaagga ctttctcctc
     1321 agaccaagaa gtcataagtc tcgagttaag ggatttttgc gattgaaaat ggcctatatg
     1381 ccaaaaaatg gaggtcaaga tgaagaaaac agtgaccaga gggatgacat ggagcatgga
     1441 tgggaagttg ttgactcaaa tgactcggct tctcagcacc aagaggaact tcctcctcct
     1501 cctctgcctc ccgggtggga agaaaaagtg gacaatttag gccgaactta ctatgtcaac
     1561 cacaacaacc ggaccactca gtggcacaga ccaagcctga tggacgtgtc ctcggagtcg
     1621 gacaataaca tcagacagat caaccaggag gcagcacacc ggcgcttccg ctcccgcagg
     1681 cacatcagcg aagacttgga gcccgagccc tcggagggcg gggatgtccc cgagccttgg
     1741 gagaccattt cagaggaagt gaatatcgct ggagactctc tcggtctggc tctgccccca
     1801 ccaccggcct ccccaggatc tcggaccagc cctcaggagc tgtcagagga actaagcaga
     1861 aggcttcaga tcactccaga ctccaatggg gaacagttca gctctttgat tcaaagagaa
     1921 ccctcctcaa ggttgaggtc atgcagtgtc accgacgcag ttgcagaaca gggccatcta
     1981 ccaccgccca gtgccccagc tgggagagcg cgttcatcaa ctgtcacggg tggtgaggaa
     2041 ccaacgccat cagtggccta tgtacatact acgccgggtc tgccttcagg ctgggaagaa
     2101 agaaaagatg ctaaggggcg cacatactat gtcaatcata acaatcgaac cacaacttgg
     2161 actcgaccta tcatgcagct tgcagaagat ggtgcgtccg gatcagccac aaacagtaac
     2221 aaccatctaa tcgagcctca gatccgccgg cctcgtagcc tcagctcgcc aacagtaact
     2281 ttatctgccc cgctggaggg tgccaaggac tcacccgtac gtcgggctgt gaaagacacc
     2341 ctttccaacc cacagtcccc acagccatca ccttacaact cccccaaacc acaacacaaa
     2401 gtcacacaga gcttcttgcc acccggctgg gaaatgagga tagcgccaaa cggccggccc
     2461 ttcttcattg atcataacac aaagactaca acctgggaag atccacgttt gaaatttcca
     2521 gtacatatgc ggtcaaagac atctttaaac cccaatgacc ttggccccct tcctcctggc
     2581 tgggaagaaa gaattcactt ggatggccga acgttttata ttgatcataa tagcaaaatt
     2641 actcagtggg aagacccaag actgcagaac ccagctatta ctggtccggc tgtcccttac
     2701 tccagagaat ttaagcagaa atatgactac ttcaggaaga aattaaagaa acctgctgat
     2761 atccccaata ggtttgaaat gaaacttcac agaaataaca tatttgaaga gtcctatcgg
     2821 agaattatgt ccgtgaaaag accagatgtc ctaaaagcta gactgtggat tgagtttgaa
     2881 tcagagaaag gtcttgacta tgggggtgtg gccagagaat ggttcttctt actgtccaaa
     2941 gagatgttca acccctacta cggcctcttt gagtactctg ccacggacaa ctacaccctt
     3001 cagatcaacc ctaattcagg cctctgtaat gaggatcatt tgtcctactt cacttttatt
     3061 ggaagagttg ctggtctggc cgtatttcat gggaagctct tagatggttt cttcattaga
     3121 ccattttaca agatgatgtt gggaaagcag ataaccctga atgacatgga atctgtggat
     3181 agtgaatatt acaactcttt gaaatggatc ctggagaatg accctactga gctggacctc
     3241 atgttctgca tagacgaaga aaactttgga cagacatatc aagtggattt gaagcccaat
     3301 gggtcagaaa taatggtcac aaatgaaaac aaaagggaat atatcgactt agtcatccag
     3361 tggagatttg tgaacagggt ccagaagcag atgaacgcct tcttggaggg attcacagaa
     3421 ctacttccta ttgatttgat taaaattttt gatgaaaatg agctggagtt gctcatgtgc
     3481 ggcctcggtg atgtggatgt gaatgactgg agacagcatt ctatttacaa gaacggctac
     3541 tgcccaaacc accccgtcat tcagtggttc tggaaggctg tgctactcat ggacgccgaa
     3601 aagcgtatcc ggttactgca gtttgtcaca gggacatcgc gagtacctat gaatggattt
     3661 gccgaacttt atggttccaa tggtcctcag ctatttacaa tagagcaatg gggcagtcct
     3721 gagaaactgc ccagagctca cacaagtttt aatcgccttg acttacctcc atatgaaacc
     3781 tttgaagatt tacgagagaa acttctcatg gccgtggaaa atgctcaagg atttgaaggg
     3841 gtggattaag cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga
     3901 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     3961 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     4021 agccatgacc cagtcacgta gcgatagcgg agtgtataat tcttgaagac gaaagggcct
     4081 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     4141 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4201 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4261 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4321 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     4381 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4441 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4501 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4561 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4621 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4681 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4741 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4801 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4861 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4921 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4981 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5041 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5101 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5161 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5221 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5281 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5341 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5401 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     5461 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5521 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5581 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5641 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5701 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5761 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5821 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5881 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5941 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     6001 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     6061 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     6121 cataaattcc gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc
     6181 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     6241 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     6301 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     6361 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     6421 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     6481 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     6541 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     6601 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     6661 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     6721 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     6781 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     6841 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     6901 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     6961 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     7021 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     7081 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     7141 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccacgctagc
     7201 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     7261 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     7321 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     7381 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgga ttcactggcc
     7441 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     7501 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     7561 caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg
     7621 gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca
     7681 aactggcaga tgcacggtta cgatgcgccc atctacacca acgtgaccta tcccattacg
     7741 gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat
     7801 gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttggaatt
     7861 agcttatcga ctgcacggtg caccaatgct tctggcgtca g
//
