LOCUS       sequence-372421-        6120 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express GST-tagged NEDD4L HECT domain in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6120)
  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 6120)
  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_HECT
FEATURES             Location/Qualifiers
     source          1..6120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(124..129,130..145,146..152)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    160..176
                     /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             199..852
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     810..832
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             859..882
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2111..2133)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2278..2296)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2364..2468
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2469..2537,2538..3329)
                     /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(2687..2706)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3500..4088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3989..4008
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4242..4259
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4332..4409
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4410..5492
                     /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(4429..4448)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5541..5546,5547..5564,5565..5571)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5579..5595
                     /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     5603..5619
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5603..5619
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5615..6120,1..22)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5635..5652)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5635..5651)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5644..5666)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg cgcactcccg
       61 ttctggataa tgttttttgc gccgacatca taacggttct ggcaaatatt ctgaaatgag
      121 ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc
      181 acacaggaaa cagtattcat gtcccctata ctaggttatt ggaaaattaa gggccttgtg
      241 caacccactc gacttctttt ggaatatctt gaagaaaaat atgaagagca tttgtatgag
      301 cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat
      361 cttccttatt atattgatgg tgatgttaaa ttaacacagt ctatggccat catacgttat
      421 atagctgaca agcacaacat gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg
      481 cttgaaggag cggttttgga tattagatac ggtgtttcga gaattgcata tagtaaagac
      541 tttgaaactc tcaaagttga ttttcttagc aagctacctg aaatgctgaa aatgttcgaa
      601 gatcgtttat gtcataaaac atatttaaat ggtgatcatg taacccatcc tgacttcatg
      661 ttgtatgacg ctcttgatgt tgttttatac atggacccaa tgtgcctgga tgcgttccca
      721 aaattagttt gttttaaaaa acgtattgaa gctatcccac aaattgataa gtacttgaaa
      781 tccagcaagt atatagcatg gcctttgcag ggctggcaag ccacgtttgg tggtggcgac
      841 catcctccaa aatcggatct ggaagttctg ttccaggggc ccctgggatc cagagaattt
      901 aagcagaaat atgactactt caggaagaaa ttaaagaaac ctgctgatat ccccaatagg
      961 tttgaaatga aacttcacag aaataacata tttgaagagt cctatcggag aattatgtcc
     1021 gtgaaaagac cagatgtcct aaaagctaga ctgtggattg agtttgaatc agagaaaggt
     1081 cttgactatg ggggtgtggc cagagaatgg ttcttcttac tgtccaaaga gatgttcaac
     1141 ccctactacg gcctctttga gtactctgcc acggacaact acacccttca gatcaaccct
     1201 aattcaggcc tctgtaatga ggatcatttg tcctacttca cttttattgg aagagttgct
     1261 ggtctggccg tatttcatgg gaagctctta gatggtttct tcattagacc attttacaag
     1321 atgatgttgg gaaagcagat aaccctgaat gacatggaat ctgtggatag tgaatattac
     1381 aactctttga aatggatcct ggagaatgac cctactgagc tggacctcat gttctgcata
     1441 gacgaagaaa actttggaca gacatatcaa gtggatttga agcccaatgg gtcagaaata
     1501 atggtcacaa atgaaaacaa aagggaatat atcgacttag tcatccagtg gagatttgtg
     1561 aacagggtcc agaagcagat gaacgccttc ttggagggat tcacagaact acttcctatt
     1621 gatttgatta aaatttttga tgaaaatgag ctggagttgc tcatgtgcgg cctcggtgat
     1681 gtggatgtga atgactggag acagcattct atttacaaga acggctactg cccaaaccac
     1741 cccgtcattc agtggttctg gaaggctgtg ctactcatgg acgccgaaaa gcgtatccgg
     1801 ttactgcagt ttgtcacagg gacatcgcga gtacctatga atggatttgc cgaactttat
     1861 ggttccaatg gtcctcagct atttacaata gagcaatggg gcagtcctga gaaactgccc
     1921 agagctcaca catgctttaa tcgccttgac ttacctccat atgaaacctt tgaagattta
     1981 cgagagaaac ttctcatggc cgtggaaaat gctcaaggat ttgaaggggt ggattaagcg
     2041 gccgcctcga gcggccgcat cgtgactgac tgacgatctg cctcgcgcgt ttcggtgatg
     2101 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     2161 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     2221 cagccatgac ccagtcacgt agcgatagcg gagtgtataa ttcttgaaga cgaaagggcc
     2281 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     2341 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2401 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2461 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2521 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2581 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2641 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2701 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2761 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     2821 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     2881 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     2941 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3001 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3061 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3121 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3181 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3241 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3301 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3361 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3421 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3481 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3541 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3601 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     3661 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3721 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3781 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3841 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3901 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3961 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4021 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4081 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4141 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4201 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4261 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4321 gcataaattc cgacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc
     4381 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     4441 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     4501 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     4561 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     4621 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     4681 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     4741 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     4801 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     4861 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     4921 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     4981 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     5041 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     5101 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     5161 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     5221 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     5281 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     5341 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accacgctag
     5401 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     5461 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc
     5521 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     5581 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgg attcactggc
     5641 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     5701 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     5761 ccaacagttg cgcagcctga atggcgaatg gcgctttgcc tggtttccgg caccagaagc
     5821 ggtgccggaa agctggctgg agtgcgatct tcctgaggcc gatactgtcg tcgtcccctc
     5881 aaactggcag atgcacggtt acgatgcgcc catctacacc aacgtgacct atcccattac
     5941 ggtcaatccg ccgtttgttc ccacggagaa tccgacgggt tgttactcgc tcacatttaa
     6001 tgttgatgaa agctggctac aggaaggcca gacgcgaatt atttttgatg gcgttggaat
     6061 tagcttatcg actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt
//
