LOCUS       sequence-338242-        8034 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses NEDD4L with inactivating mutation in first, second and 
            fourth WW domain, but intact third WW domain, in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8034)
  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 8034)
  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∆WW1,2,4
FEATURES             Location/Qualifiers
     source          1..8034
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        224..603
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        604..815
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     766..786
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     812..836
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          976..1108
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1091..1110
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1153..1171
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1194..1217
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    3835..3956
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3846..3865)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3900..3919
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      4142..4597
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4229..4248)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4439..4460
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4714..5071
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4714..4905
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4737..4757)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4922..5057
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5122..5916
                     /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(5176..5195)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5786..5805
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5980..6028
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(6069..6088)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6188..6210
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6248..6266)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6334..6438
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6439..6507,6508..7299)
                     /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(6657..6676)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7470..8034,1..24)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7959..7978
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
       61 ttttgctcac atggctcgac agatcttcaa tattggccat tagccatatt attcattggt
      121 tatatagcat aaatcaatat tggctattgg ccattgcata cgttgtatct atatcataat
      181 atgtacattt atattggctc atgtccaata tgaccgccat gttggcattg attattgact
      241 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      301 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      361 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      421 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatccca
      481 agtccgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      541 atgaccttac gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      601 atggtgatgc ggttttggca gtacaccaat gggcgtggat agcggtttga ctcacgggga
      661 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      721 gactttccaa aatgtcgtaa caactgcgat cgcccgcccc gttgacgcaa atgggcggta
      781 ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcacta
      841 gaagctttat tgcggtagtt tatcacagtt aaattgctaa cgcagtcagt gcttctgaca
      901 caacagtctc gaacttaagc tgcagtgact ctcttaaggt agccttgcag aagttggtcg
      961 tgaggcactg ggcaggtaag tatcaaggtt acaagacagg tttaaggaga ccaatagaaa
     1021 ctgggcttgt cgagacagag aagactcttg cgtttctgat aggcacctat tggtcttact
     1081 gacatccact ttgcctttct ctccacaggt gtccactccc agttcaatta cagctcttaa
     1141 ggctagagta cttaatacga ctcactatag gctagcctcg agaattcgcc atggattaca
     1201 aggatgacga cgataagggc catatggaac gaccctatac atttaaggac tttctcctca
     1261 gaccaagaag tcataagtct cgagttaagg gatttttgcg attgaaaatg gcctatatgc
     1321 caaaaaatgg aggtcaagat gaagaaaaca gtgaccagag ggatgacatg gagcatggat
     1381 gggaagttgt tgactcaaat gactcggctt ctcagcacca agaggaactt cctcctcctc
     1441 ctctgcctcc cgggtgggaa gaaaaagtgg acaatttagg ccgaacttac tatgtcaacc
     1501 acaacaaccg gaccactcag gctcacagac caagcctgat ggacgtgtcc tcggagtcgg
     1561 acaataacat cagacagatc aaccaggagg cagcacaccg gcgcttccgc tcccgcaggc
     1621 acatcagcga agacttggag cccgagccct cggagggcgg ggatgtcccc gagccttggg
     1681 agaccatttc agaggaagtg aatatcgctg gagactctct cggtctggct ctgcccccac
     1741 caccggcctc cccaggatct cggaccagcc ctcaggagct gtcagaggaa ctaagcagaa
     1801 ggcttcagat cactccagac tccaatgggg aacagttcag ctctttgatt caaagagaac
     1861 cctcctcaag gttgaggtca tgcagtgtca ccgacgcagt tgcagaacag ggccatctac
     1921 caccgcccag tgccccagct gggagagcgc gttcatcaac tgtcacgggt ggtgaggaac
     1981 caacgccatc agtggcctat gtacatacca cgccgggtct gccttcaggc tgggaagaaa
     2041 gaaaagatgc taaggggcgc acatactatg tcaatcataa caatcgaacc acaactgcca
     2101 ctcgacctat catgcagctt gcagaagatg gtgcgtccgg atcagccaca aacagtaaca
     2161 accatctaat cgagcctcag atccgccggc ctcgtagcct cagctcgcca acagtaactt
     2221 tatctgcccc gctggagggt gccaaggact cacccgtacg tcgggctgtg aaagacaccc
     2281 tttccaaccc acagtcccca cagccatcac cttacaactc ccccaaacca caacacaaag
     2341 tcacacagag cttcttgcca cccggctggg aaatgaggat agcgccaaac ggccggccct
     2401 tcttcattga tcataacaca aagactacaa cctgggaaga tccacgtttg aaatttccag
     2461 tacatatgcg gtcaaagaca tctttaaacc ccaatgacct tggccccctt cctcctggct
     2521 gggaagaaag aattcacttg gatggccgaa cgttttatat tgatcataat agcaaaatta
     2581 ctcaggccga agacccaaga ctgcagaacc cagctattac tggtccggct gtcccttact
     2641 ccagagaatt taagcagaaa tatgactact tcaggaagaa attaaagaaa cctgctgata
     2701 tccccaatag gtttgaaatg aaacttcaca gaaataacat atttgaagag tcctatcgga
     2761 gaattatgtc cgtgaaaaga ccagatgtcc taaaagctag actgtggatt gagtttgaat
     2821 cagagaaagg tcttgactat gggggtgtgg ccagagaatg gttcttctta ctgtccaaag
     2881 agatgttcaa cccctactac ggcctctttg agtactctgc cacggacaac tacacccttc
     2941 agatcaaccc taattcaggc ctctgtaatg aggatcattt gtcctacttc acttttattg
     3001 gaagagttgc tggtctggcc gtatttcatg ggaagctctt agatggtttc ttcattagac
     3061 cattttacaa gatgatgttg ggaaagcaga taaccctgaa tgacatggaa tctgtggata
     3121 gtgaatatta caactctttg aaatggatcc tggagaatga ccctactgag ctggacctca
     3181 tgttctgcat agacgaagaa aactttggac agacatatca agtggatttg aagcccaatg
     3241 ggtcagaaat aatggtcaca aatgaaaaca aaagggaata tatcgactta gtcatccagt
     3301 ggagatttgt gaacagggtc cagaagcaga tgaacgcctt cttggaggga ttcacagaac
     3361 tacttcctat tgatttgatt aaaatttttg acgaaaatga gctggagttg ctcatgtgcg
     3421 gcctcggtga tgtggatgtg aatgactgga gacagcattc tatttacaag aacggctact
     3481 gcccaaacca ccccgtcatt cagtggttct ggaaggctgt gctactcatg gacgccgaaa
     3541 agcgtatccg gttactgcag tttgtcacag ggacatcgcg agtacctatg aatggatttg
     3601 ccgaacttta tggttccaat ggtcctcagc tgtttacaat agagcaatgg ggcagtcctg
     3661 agaaactgcc cagagctcac acatgcttta atcgccttga cttacctcca tatgaaacct
     3721 ttgaagattt acgagagaaa cttctcatgg ccgtggaaaa tgctcaagga tttgaagggg
     3781 tggattaagc ggccgcttcc ctttagtgag ggttaatgct tcgagcagac atgataagat
     3841 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     3901 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     3961 acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa
     4021 gcaagtaaaa cctctacaaa tgtggtaaaa tccgataagg atcgatccgg gctggcgtaa
     4081 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     4141 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4201 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4261 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4321 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     4381 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4441 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4501 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4561 aacgcgaatt ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta
     4621 cgcatctgtg cggtatttca caccgcatac gcggatctgc gcagcaccat ggcctgaaat
     4681 aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa
     4741 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4801 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     4861 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     4921 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4981 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     5041 aggctttttt ggaggcctag gcttttgcaa aaagcttgat tcttctgaca caacagtctc
     5101 gaacttaagg ctagagccac catgattgaa caagatggat tgcacgcagg ttctccggcc
     5161 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     5221 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     5281 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     5341 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     5401 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     5461 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     5521 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     5581 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5641 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     5701 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5761 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5821 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5881 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5941 ccgaccaagc gacgcccaac ctgccatcac gatggccgca ataaaatatc tttattttca
     6001 ttacatctgt gtgttggttt tttgtgtgaa tcgatagcga taaggatacg cgtatggtgc
     6061 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     6121 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     6181 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     6241 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     6301 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     6361 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6421 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6481 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6541 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6601 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6661 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6721 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6781 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6841 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6901 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6961 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     7021 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     7081 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     7141 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     7201 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     7261 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7321 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7381 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7441 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7501 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     7561 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     7621 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7681 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7741 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7801 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7861 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7921 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7981 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcg
//
