LOCUS       sequence-372432-        8345 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Express the catalytically inactive NEDD4L FL in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8345)
  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 8345)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_C942S_pCDNA3.1(+)
FEATURES             Location/Qualifiers
     source          1..8345
                     /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(3936..3953)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3942..4166
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4212..4640
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4299..4318)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4509..4530
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4649..4669)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4654..4983
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4834..4969
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4896..4915
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5050..5844
                     /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(5104..5123)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5714..5733
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6018..6139
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6055..6074)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6109..6128
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6188..6204)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6188..6204)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6201..6223)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6212..6228
                     /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(6236..6242,6243..6260,6261..6266))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6281..6302
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6419..6436)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6590..7178)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6670..6689)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7349..8140,8141..8209))
                     /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     7972..7991
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8210..8314)
                     /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 cttgcagaag atggtgcgtc cggatcagcc acaaacagta acaaccatct aatcgagcct
     2281 cagatccgcc ggcctcgtag cctcagctcg ccaacagtaa ctttatctgc cccgctggag
     2341 ggtgccaagg actcacccgt acgtcgggct gtgaaagaca ccctttccaa cccacagtcc
     2401 ccacagccat caccttacaa ctcccccaaa ccacaacaca aagtcacaca gagcttcttg
     2461 ccacccggct gggaaatgag gatagcgcca aacggccggc ccttcttcat tgatcataac
     2521 acaaagacta caacctggga agatccacgt ttgaaatttc cagtacatat gcggtcaaag
     2581 acatctttaa accccaatga ccttggcccc cttcctcctg gctgggaaga aagaattcac
     2641 ttggatggcc gaacgtttta tattgatcat aatagcaaaa ttactcagtg ggaagaccca
     2701 agactgcaga acccagctat tactggtccg gctgtccctt actccagaga atttaagcag
     2761 aaatatgact acttcaggaa gaaattaaag aaacctgctg atatccccaa taggtttgaa
     2821 atgaaacttc acagaaataa catatttgaa gagtcctatc ggagaattat gtccgtgaaa
     2881 agaccagatg tcctaaaagc tagactgtgg attgagtttg aatcagagaa aggtcttgac
     2941 tatgggggtg tggccagaga atggttcttc ttactgtcca aagagatgtt caacccctac
     3001 tacggcctct ttgagtactc tgccacggac aactacaccc ttcagatcaa ccctaattca
     3061 ggcctctgta atgaggatca tttgtcctac ttcactttta ttggaagagt tgctggtctg
     3121 gccgtatttc atgggaagct cttagatggt ttcttcatta gaccatttta caagatgatg
     3181 ttgggaaagc agataaccct gaatgacatg gaatctgtgg atagtgaata ttacaactct
     3241 ttgaaatgga tcctggagaa tgaccctact gagctggacc tcatgttctg catagacgaa
     3301 gaaaactttg gacagacata tcaagtggat ttgaagccca atgggtcaga aataatggtc
     3361 acaaatgaaa acaaaaggga atatatcgac ttagtcatcc agtggagatt tgtgaacagg
     3421 gtccagaagc agatgaacgc cttcttggag ggattcacag aactacttcc tattgatttg
     3481 attaaaattt ttgatgaaaa tgagctggag ttgctcatgt gcggcctcgg tgatgtggat
     3541 gtgaatgact ggagacagca ttctatttac aagaacggct actgcccaaa ccaccccgtc
     3601 attcagtggt tctggaaggc tgtgctactc atggacgccg aaaagcgtat ccggttactg
     3661 cagtttgtca cagggacatc gcgagtacct atgaatggat ttgccgaact ttatggttcc
     3721 aatggtcctc agctatttac aatagagcaa tggggcagtc ctgagaaact gcccagagct
     3781 cacacatctt ttaatcgcct tgacttacct ccatatgaaa cctttgaaga tttacgagag
     3841 aaacttctca tggccgtgga aaatgctcaa ggatttgaag gggtggatta agcggccgct
     3901 cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     3961 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     4021 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     4081 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     4141 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     4201 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4261 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4321 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4381 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4441 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4501 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4561 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4621 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     4681 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     4741 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4801 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4861 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4921 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4981 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
     5041 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     5101 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     5161 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     5221 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     5281 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     5341 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     5401 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     5461 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     5521 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     5581 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     5641 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     5701 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     5761 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     5821 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     5881 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5941 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     6001 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     6061 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     6121 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     6181 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     6241 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     6301 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     6361 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     6421 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     6481 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     6541 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6601 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6661 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6721 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6781 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6841 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6901 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6961 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     7021 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     7081 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     7141 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     7201 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     7261 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     7321 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     7381 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     7441 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     7501 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     7561 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7621 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7681 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7741 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7801 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7861 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7921 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7981 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     8041 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     8101 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     8161 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     8221 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     8281 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     8341 acgtc
//
