LOCUS       sequence-372425-        7877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express his-tagged NEDD4L FL with removal of the linker between the 
            WW1 and WW2 domains in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7877)
  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 7877)
  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-1,2-linker_pcDNA3.1(+)
FEATURES             Location/Qualifiers
     source          1..7877
                     /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(3468..3485)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3474..3698
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3744..4172
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3831..3850)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4041..4062
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4181..4201)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4186..4515
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4366..4501
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4428..4447
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4582..5376
                     /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(4636..4655)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5246..5265
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5550..5671
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5587..5606)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5641..5660
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5720..5736)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5720..5736)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5733..5755)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5744..5760
                     /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(5768..5774,5775..5792,5793..5798))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5813..5834
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5951..5968)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6122..6710)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6202..6221)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6881..7672,7673..7741))
                     /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     7504..7523
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7742..7846)
                     /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 gactacgccg ggtctgcctt caggctggga agaaagaaaa
     1681 gatgctaagg ggcgcacata ctatgtcaat cataacaatc gaaccacaac ttggactcga
     1741 cctatcatgc agcttgcaga agatggtgcg tccggatcag ccacaaacag taacaaccat
     1801 ctaatcgagc ctcagatccg ccggcctcgt agcctcagct cgccaacagt aactttatct
     1861 gccccgctgg agggtgccaa ggactcaccc gtacgtcggg ctgtgaaaga caccctttcc
     1921 aacccacagt ccccacagcc atcaccttac aactccccca aaccacaaca caaagtcaca
     1981 cagagcttct tgccacccgg ctgggaaatg aggatagcgc caaacggccg gcccttcttc
     2041 attgatcata acacaaagac tacaacctgg gaagatccac gtttgaaatt tccagtacat
     2101 atgcggtcaa agacatcttt aaaccccaat gaccttggcc cccttcctcc tggctgggaa
     2161 gaaagaattc acttggatgg ccgaacgttt tatattgatc ataatagcaa aattactcag
     2221 tgggaagacc caagactgca gaacccagct attactggtc cggctgtccc ttactccaga
     2281 gaatttaagc agaaatatga ctacttcagg aagaaattaa agaaacctgc tgatatcccc
     2341 aataggtttg aaatgaaact tcacagaaat aacatatttg aagagtccta tcggagaatt
     2401 atgtccgtga aaagaccaga tgtcctaaaa gctagactgt ggattgagtt tgaatcagag
     2461 aaaggtcttg actatggggg tgtggccaga gaatggttct tcttactgtc caaagagatg
     2521 ttcaacccct actacggcct ctttgagtac tctgccacgg acaactacac ccttcagatc
     2581 aaccctaatt caggcctctg taatgaggat catttgtcct acttcacttt tattggaaga
     2641 gttgctggtc tggccgtatt tcatgggaag ctcttagatg gtttcttcat tagaccattt
     2701 tacaagatga tgttgggaaa gcagataacc ctgaatgaca tggaatctgt ggatagtgaa
     2761 tattacaact ctttgaaatg gatcctggag aatgacccta ctgagctgga cctcatgttc
     2821 tgcatagacg aagaaaactt tggacagaca tatcaagtgg atttgaagcc caatgggtca
     2881 gaaataatgg tcacaaatga aaacaaaagg gaatatatcg acttagtcat ccagtggaga
     2941 tttgtgaaca gggtccagaa gcagatgaac gccttcttgg agggattcac agaactactt
     3001 cctattgatt tgattaaaat ttttgatgaa aatgagctgg agttgctcat gtgcggcctc
     3061 ggtgatgtgg atgtgaatga ctggagacag cattctattt acaagaacgg ctactgccca
     3121 aaccaccccg tcattcagtg gttctggaag gctgtgctac tcatggacgc cgaaaagcgt
     3181 atccggttac tgcagtttgt cacagggaca tcgcgagtac ctatgaatgg atttgccgaa
     3241 ctttatggtt ccaatggtcc tcagctattt acaatagagc aatggggcag tcctgagaaa
     3301 ctgcccagag ctcacacatg ctttaatcgc cttgacttac ctccatatga aacctttgaa
     3361 gatttacgag agaaacttct catggccgtg gaaaatgctc aaggatttga aggggtggat
     3421 taagcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     3481 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3541 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3601 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3661 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3721 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3781 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3841 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3901 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3961 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4021 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4081 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4141 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     4201 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4261 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4321 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4381 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4441 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4501 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     4561 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4621 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4681 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     4741 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     4801 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4861 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4921 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4981 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     5041 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5101 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     5161 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5221 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5281 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5341 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5401 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     5461 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5521 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     5581 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5641 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     5701 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5761 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5821 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5881 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5941 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     6001 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     6061 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     6121 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     6181 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6241 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6301 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6361 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6421 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6481 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6541 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6601 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6661 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6721 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6781 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6841 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6901 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6961 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     7021 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     7081 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     7141 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     7201 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     7261 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     7321 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7381 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7441 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7501 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7561 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7621 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7681 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7741 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7801 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7861 aagtgccacc tgacgtc
//
