LOCUS       sequence-372426-        8114 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express his-tagged NEDD4L FL with removal of the linker between the 
            WW2 and WW3 domains in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8114)
  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 8114)
  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: His-Precission-NEDD4L_Delta-2,3-linker_pcDNA3.1(+)
FEATURES             Location/Qualifiers
     source          1..8114
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             904..921
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             925..948
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(3705..3722)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3711..3935
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3981..4409
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4068..4087)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4278..4299
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4418..4438)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4423..4752
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4603..4738
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4665..4684
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4819..5613
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4873..4892)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5483..5502
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5787..5908
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5824..5843)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5878..5897
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5957..5973)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5957..5973)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5970..5992)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5981..5997
                     /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)."
     promoter        complement(join(6005..6011,6012..6029,6030..6035))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6050..6071
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6188..6205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6359..6947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6439..6458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7118..7909,7910..7978))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7741..7760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7979..8083)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 atgcaccacc accaccacca cggcctggag gtgctgttcc agggcccagt acgcgccggc
      961 tccatggcga ccgggctcgg ggagccggtc tatggacttt ccgaagacga gggagagtcc
     1021 cgtattctca gagtaaaagt tgtttctgga attgatctcg ccaaaaagga catctttgga
     1081 gccagtgatc cgtatgtgaa actttcattg tacgtagcgg atgagaatag agaacttgct
     1141 ttggtccaga caaaaacaat taaaaagaca ctgaacccaa aatggaatga agaattttat
     1201 ttcagggtaa acccatctaa tcacagactc ctatttgaag tatttgacga aaatagactg
     1261 acacgagacg acttcctggg ccaggtggac gtgcccctta gtcaccttcc gacagaagat
     1321 ccaaccatgg agcgacccta tacatttaag gactttctcc tcagaccaag aagtcataag
     1381 tctcgagtta agggattttt gcgattgaaa atggcctata tgccaaaaaa tggaggtcaa
     1441 gatgaagaaa acagtgacca gagggatgac atggagcatg gatgggaagt tgttgactca
     1501 aatgactcgg cttctcagca ccaagaggaa cttcctcctc ctcctctgcc tcccgggtgg
     1561 gaagaaaaag tggacaattt aggccgaact tactatgtca accacaacaa ccggaccact
     1621 cagtggcaca gaccaagcct gatggacgtg tcctcggagt cggacaataa catcagacag
     1681 atcaaccagg aggcagcaca ccggcgcttc cgctcccgca ggcacatcag cgaagacttg
     1741 gagcccgagc cctcggaggg cggggatgtc cccgagcctt gggagaccat ttcagaggaa
     1801 gtgaatatcg ctggagactc tctcggtctg gctctgcccc caccaccggc ctccccagga
     1861 tctcggacca gccctcagga gctgtcagag gaactaagca gaaggcttca gatcactcca
     1921 gactccaatg gggaacagtt cagctctttg attcaaagag aaccctcctc aaggttgagg
     1981 tcatgcagtg tcaccgacgc agttgcagaa cagggccatc taccaccgcc cagtgcccca
     2041 gctgggagag cgcgttcatc aactgtcacg ggtggtgagg aaccaacgcc atcagtggcc
     2101 tatgtacata ctacgccggg tctgccttca ggctgggaag aaagaaaaga tgctaagggg
     2161 cgcacatact atgtcaatca taacaatcga accacaactt ggactcgacc tatcatgcag
     2221 agcttcttgc cacccggctg ggaaatgagg atagcgccaa acggccggcc cttcttcatt
     2281 gatcataaca caaagactac aacctgggaa gatccacgtt tgaaatttcc agtacatatg
     2341 cggtcaaaga catctttaaa ccccaatgac cttggccccc ttcctcctgg ctgggaagaa
     2401 agaattcact tggatggccg aacgttttat attgatcata atagcaaaat tactcagtgg
     2461 gaagacccaa gactgcagaa cccagctatt actggtccgg ctgtccctta ctccagagaa
     2521 tttaagcaga aatatgacta cttcaggaag aaattaaaga aacctgctga tatccccaat
     2581 aggtttgaaa tgaaacttca cagaaataac atatttgaag agtcctatcg gagaattatg
     2641 tccgtgaaaa gaccagatgt cctaaaagct agactgtgga ttgagtttga atcagagaaa
     2701 ggtcttgact atgggggtgt ggccagagaa tggttcttct tactgtccaa agagatgttc
     2761 aacccctact acggcctctt tgagtactct gccacggaca actacaccct tcagatcaac
     2821 cctaattcag gcctctgtaa tgaggatcat ttgtcctact tcacttttat tggaagagtt
     2881 gctggtctgg ccgtatttca tgggaagctc ttagatggtt tcttcattag accattttac
     2941 aagatgatgt tgggaaagca gataaccctg aatgacatgg aatctgtgga tagtgaatat
     3001 tacaactctt tgaaatggat cctggagaat gaccctactg agctggacct catgttctgc
     3061 atagacgaag aaaactttgg acagacatat caagtggatt tgaagcccaa tgggtcagaa
     3121 ataatggtca caaatgaaaa caaaagggaa tatatcgact tagtcatcca gtggagattt
     3181 gtgaacaggg tccagaagca gatgaacgcc ttcttggagg gattcacaga actacttcct
     3241 attgatttga ttaaaatttt tgatgaaaat gagctggagt tgctcatgtg cggcctcggt
     3301 gatgtggatg tgaatgactg gagacagcat tctatttaca agaacggcta ctgcccaaac
     3361 caccccgtca ttcagtggtt ctggaaggct gtgctactca tggacgccga aaagcgtatc
     3421 cggttactgc agtttgtcac agggacatcg cgagtaccta tgaatggatt tgccgaactt
     3481 tatggttcca atggtcctca gctatttaca atagagcaat ggggcagtcc tgagaaactg
     3541 cccagagctc acacatgctt taatcgcctt gacttacctc catatgaaac ctttgaagat
     3601 ttacgagaga aacttctcat ggccgtggaa aatgctcaag gatttgaagg ggtggattaa
     3661 gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     3721 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3781 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3841 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3901 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     3961 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4021 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     4081 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4141 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     4201 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     4261 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     4321 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     4381 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     4441 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4501 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4561 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     4621 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4681 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4741 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     4801 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     4861 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     4921 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     4981 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     5041 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     5101 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     5161 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     5221 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     5281 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     5341 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     5401 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     5461 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     5521 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     5581 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     5641 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     5701 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     5761 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     5821 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5881 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     5941 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     6001 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     6061 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     6121 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     6181 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     6241 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     6301 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     6361 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     6421 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     6481 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6541 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6601 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6661 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6721 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6781 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6841 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6901 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6961 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     7021 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     7081 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     7141 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     7201 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     7261 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     7321 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     7381 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     7441 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     7501 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7561 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7621 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7681 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7741 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7801 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7861 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7921 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7981 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     8041 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     8101 tgccacctga cgtc
//
