LOCUS       sequence-372420-        6860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express his-tagged NEDD4L W3-W4-HECT in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6860)
  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 6860)
  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 (492-975)_pcDNA3.1(+)
FEATURES             Location/Qualifiers
     source          1..6860
                     /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             907..924
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             928..951
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2451..2468)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2457..2681
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2727..3155
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2814..2833)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3024..3045
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3164..3184)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3169..3498
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3349..3484
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3411..3430
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3565..4359
                     /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(3619..3638)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4229..4248
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4533..4654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4570..4589)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4624..4643
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4703..4719)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4703..4719)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4716..4738)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4727..4743
                     /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(4751..4757,4758..4775,4776..4781))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4796..4817
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4934..4951)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5105..5693)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5185..5204)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5864..6655,6656..6724))
                     /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     6487..6506
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6725..6829)
                     /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 atggggcacc accaccacca ccacggcctg gaggtgctgt tccagggccc acacaaagtc
      961 acacagagct tcttgccacc cggctgggaa atgaggatag cgccaaacgg ccggcccttc
     1021 ttcattgatc ataacacaaa gactacaacc tgggaagatc cacgtttgaa atttccagta
     1081 catatgcggt caaagacatc tttaaacccc aatgaccttg gcccccttcc tcctggctgg
     1141 gaagaaagaa ttcacttgga tggccgaacg ttttatattg atcataatag caaaattact
     1201 cagtgggaag acccaagact gcagaaccca gctattactg gtccggctgt cccttactcc
     1261 agagaattta agcagaaata tgactacttc aggaagaaat taaagaaacc tgctgatatc
     1321 cccaataggt ttgaaatgaa acttcacaga aataacatat ttgaagagtc ctatcggaga
     1381 attatgtccg tgaaaagacc agatgtccta aaagctagac tgtggattga gtttgaatca
     1441 gagaaaggtc ttgactatgg gggtgtggcc agagaatggt tcttcttact gtccaaagag
     1501 atgttcaacc cctactacgg cctctttgag tactctgcca cggacaacta cacccttcag
     1561 atcaacccta attcaggcct ctgtaatgag gatcatttgt cctacttcac ttttattgga
     1621 agagttgctg gtctggccgt atttcatggg aagctcttag atggtttctt cattagacca
     1681 ttttacaaga tgatgttggg aaagcagata accctgaatg acatggaatc tgtggatagt
     1741 gaatattaca actctttgaa atggatcctg gagaatgacc ctactgagct ggacctcatg
     1801 ttctgcatag acgaagaaaa ctttggacag acatatcaag tggatttgaa gcccaatggg
     1861 tcagaaataa tggtcacaaa tgaaaacaaa agggaatata tcgacttagt catccagtgg
     1921 agatttgtga acagggtcca gaagcagatg aacgccttct tggagggatt cacagaacta
     1981 cttcctattg atttgattaa aatttttgat gaaaatgagc tggagttgct catgtgcggc
     2041 ctcggtgatg tggatgtgaa tgactggaga cagcattcta tttacaagaa cggctactgc
     2101 ccaaaccacc ccgtcattca gtggttctgg aaggctgtgc tactcatgga cgccgaaaag
     2161 cgtatccggt tactgcagtt tgtcacaggg acatcgcgag tacctatgaa tggatttgcc
     2221 gaactttatg gttccaatgg tcctcagcta tttacaatag agcaatgggg cagtcctgag
     2281 aaactgccca gagctcacac atgctttaat cgccttgact tacctccata tgaaaccttt
     2341 gaagatttac gagagaaact tctcatggcc gtggaaaatg ctcaaggatt tgaaggggtg
     2401 gattaagcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     2461 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2521 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2581 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2641 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     2701 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     2761 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2821 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2881 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2941 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3001 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3061 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3121 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3181 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3241 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3301 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3361 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3421 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3481 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     3541 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3601 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3661 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3721 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     3781 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3841 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     3901 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     3961 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4021 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4081 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4141 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4201 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4261 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4321 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     4381 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     4441 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4501 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4561 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4621 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4681 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4741 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4801 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4861 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4921 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4981 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5041 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5101 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5161 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5221 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5281 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5341 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5401 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5461 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5521 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5581 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5641 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5701 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5761 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5821 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5881 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5941 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6001 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6061 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6121 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6181 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6241 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6301 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6361 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6421 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6481 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6541 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6601 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6661 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6721 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6781 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6841 gaaaagtgcc acctgacgtc
//
