LOCUS       sequence-329115-        8045 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses NEDD4L with inactivating mutation in third WW domain in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8045)
  AUTHORS   Rheinemann L, Thompson T, Mercenne G, Paine EL, Peterson FC, Volkman
            BF, Alam SL, Alian A, Sundquist WI
  TITLE     Interactions between AMOT PPxY Motifs and NEDD4L WW Domains Function
            in HIV-1 Release.
  JOURNAL   J Biol Chem. 2021 Jul 17:100975. doi: 10.1016/j.jbc.2021.100975.
  PUBMED    34284061
REFERENCE   2  (bases 1 to 8045)
  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. 2021 Jul 17:100975. doi: 10.1016/j.jbc.2021.100975."
COMMENT     Sequence Label: pCI-Flag-NEDD4L∆WW3
FEATURES             Location/Qualifiers
     source          1..8045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        140..519
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        520..731
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     682..702
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     728..752
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          892..1024
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1007..1026
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1069..1087
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1110..1133
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    3751..3872
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3762..3781)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3816..3835
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      4058..4513
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4145..4164)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4355..4376
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4630..4987
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4630..4821
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4653..4673)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4838..4973
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5038..5832
                     /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(5092..5111)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5702..5721
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5896..5944
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5985..6004)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6104..6126
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6164..6182)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6250..6354
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6355..6423,6424..7215)
                     /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     complement(6573..6592)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7386..7974
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7875..7894
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cttcaatatt ggccattagc catattattc attggttata tagcataaat caatattggc
       61 tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat tggctcatgt
      121 ccaatatgac cgccatgttg gcattgatta ttgactagtt attaatagta atcaattacg
      181 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      241 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      301 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      361 gcccacttgg cagtacatca agtgtatcat atcccaagtc cgccccctat tgacgtcaat
      421 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga ctttcctact
      481 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      541 accaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      601 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      661 tgcgatcgcc cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
      721 taagcagagc tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc
      781 acagttaaat tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca
      841 gtgactctct taaggtagcc ttgcagaagt tggtcgtgag gcactgggca ggtaagtatc
      901 aaggttacaa gacaggttta aggagaccaa tagaaactgg gcttgtcgag acagagaaga
      961 ctcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc ctttctctcc
     1021 acaggtgtcc actcccagtt caattacagc tcttaaggct agagtactta atacgactca
     1081 ctataggcta gcctcgagaa ttcgccatgg attacaagga tgacgacgat aagggccata
     1141 tggaacgacc ctatacattt aaggactttc tcctcagacc aagaagtcat aagtctcgag
     1201 ttaagggatt tttgcgattg aaaatggcct atatgccaaa aaatggaggt caagatgaag
     1261 aaaacagtga ccagagggat gacatggagc atggatggga agttgttgac tcaaatgact
     1321 cggcttctca gcaccaagag gaacttcctc ctcctcctct gcctcccggg tgggaagaaa
     1381 aagtggacaa tttaggccga acttactatg tcaaccacaa caaccggacc actcagtggc
     1441 acagaccaag cctgatggac gtgtcctcgg agtcggacaa taacatcaga cagatcaacc
     1501 aggaggcagc acaccggcgc ttccgctccc gcaggcacat cagcgaagac ttggagcccg
     1561 agccctcgga gggcggggat gtccccgagc cttgggagac catttcagag gaagtgaata
     1621 tcgctggaga ctctctcggt ctggctctgc ccccaccacc ggcctcccca ggatctcgga
     1681 ccagccctca ggagctgtca gaggaactaa gcagaaggct tcagatcact ccagactcca
     1741 atggggaaca gttcagctct ttgattcaaa gagaaccctc ctcaaggttg aggtcatgca
     1801 gtgtcaccga cgcagttgca gaacagggcc atctaccacc gcccagtgcc ccagctggga
     1861 gagcgcgttc atcaactgtc acgggtggtg aggaaccaac gccatcagtg gcctatgtac
     1921 ataccacgcc gggtctgcct tcaggctggg aagaaagaaa agatgctaag gggcgcacat
     1981 actatgtcaa tcataacaat cgaaccacaa cttggactcg acctatcatg cagcttgcag
     2041 aagatggtgc gtccggatca gccacaaaca gtaacaacca tctaatcgag cctcagatcc
     2101 gccggcctcg tagcctcagc tcgccaacag taactttatc tgccccgctg gagggtgcca
     2161 aggactcacc cgtacgtcgg gctgtgaaag acaccctttc caacccacag tccccacagc
     2221 catcacctta caactccccc aaaccacaac acaaagtcac acagagcttc ttgccacccg
     2281 gctgggaaat gaggatagcg ccaaacggcc ggcccttctt cattgatcat aacacaaaga
     2341 ctacaaccgc cgaagatcca cgtttgaaat ttccagtaca tatgcggtca aagacatctt
     2401 taaaccccaa tgaccttggc ccccttcctc ctggctggga agaaagaatt cacttggatg
     2461 gccgaacgtt ttatattgat cataatagca aaattactca gtgggaagac ccaagactgc
     2521 agaacccagc tattactggt ccggctgtcc cttactccag agaatttaag cagaaatatg
     2581 actacttcag gaagaaatta aagaaacctg ctgatatccc caataggttt gaaatgaaac
     2641 ttcacagaaa taacatattt gaagagtcct atcggagaat tatgtccgtg aaaagaccag
     2701 atgtcctaaa agctagactg tggattgagt ttgaatcaga gaaaggtctt gactatgggg
     2761 gtgtggccag agaatggttc ttcttactgt ccaaagagat gttcaacccc tactacggcc
     2821 tctttgagta ctctgccacg gacaactaca cccttcagat caaccctaat tcaggcctct
     2881 gtaatgagga tcatttgtcc tacttcactt ttattggaag agttgctggt ctggccgtat
     2941 ttcatgggaa gctcttagat ggtttcttca ttagaccatt ttacaagatg atgttgggaa
     3001 agcagataac cctgaatgac atggaatctg tggatagtga atattacaac tctttgaaat
     3061 ggatcctgga gaatgaccct actgagctgg acctcatgtt ctgcatagac gaagaaaact
     3121 ttggacagac atatcaagtg gatttgaagc ccaatgggtc agaaataatg gtcacaaatg
     3181 aaaacaaaag ggaatatatc gacttagtca tccagtggag atttgtgaac agggtccaga
     3241 agcagatgaa cgccttcttg gagggattca cagaactact tcctattgat ttgattaaaa
     3301 tttttgacga aaatgagctg gagttgctca tgtgcggcct cggtgatgtg gatgtgaatg
     3361 actggagaca gcattctatt tacaagaacg gctactgccc aaaccacccc gtcattcagt
     3421 ggttctggaa ggctgtgcta ctcatggacg ccgaaaagcg tatccggtta ctgcagtttg
     3481 tcacagggac atcgcgagta cctatgaatg gatttgccga actttatggt tccaatggtc
     3541 ctcagctgtt tacaatagag caatggggca gtcctgagaa actgcccaga gctcacacat
     3601 gctttaatcg ccttgactta cctccatatg aaacctttga agatttacga gagaaacttc
     3661 tcatggccgt ggaaaatgct caaggatttg aaggggtgga ttaagcggcc gcttcccttt
     3721 agtgagggtt aatgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca
     3781 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     3841 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     3901 ttcaggttca gggggagatg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     3961 gtaaaatccg ataaggatcg atccgggctg gcgtaatagc gaagaggccc gcaccgatcg
     4021 cccttcccaa cagttgcgca gcctgaatgg cgaatggacg cgccctgtag cggcgcatta
     4081 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     4141 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4201 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4261 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4321 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4381 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     4441 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     4501 acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4561 gcatacgcgg atctgcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt
     4621 aggtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     4681 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4741 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4801 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4861 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4921 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     4981 ttgcaaaaag cttgattctt ctgacacaac agtctcgaac ttaaggctag agccaccatg
     5041 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     5101 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     5161 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     5221 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     5281 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     5341 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     5401 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     5461 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     5521 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     5581 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     5641 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     5701 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     5761 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     5821 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     5881 catcacgatg gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg
     5941 tgtgaatcga tagcgataag gatacgcgta tggtgcactc tcagtacaat ctgctctgat
     6001 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     6061 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     6121 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     6181 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     6241 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     6301 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     6361 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6421 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6481 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6541 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6601 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6661 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6721 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6781 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6841 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6901 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6961 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     7021 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     7081 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     7141 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     7201 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     7261 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     7321 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     7381 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     7441 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     7501 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     7561 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7621 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7681 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7741 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7801 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7861 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7921 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7981 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catggctcga
     8041 cagat
//
