LOCUS       sequence-323610-        5092 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses NEDD4L WW3 in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5092)
  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 5092)
  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: pGEX-PP-NEDD4L-WW3(497-530)
FEATURES             Location/Qualifiers
     source          1..5092
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             939..962
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1142..1164)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1309..1327)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1395..1499
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1500..1568,1569..2360)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1718..1737)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2531..3119
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3020..3039
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3273..3290
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3363..3440
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3441..4523
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3460..3479)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    4536..4557
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4572..4577,4578..4595,4596..4602)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4610..4626
                     /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)."
     primer_bind     4634..4650
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4634..4650
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4646..5092,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4666..4683)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4666..4682)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4675..4697)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatctccgct tgaagtcctc tttcagggac
      961 cccatatgag cttcttgcca cccggctggg aaatgaggat agcgccaaac ggccggccct
     1021 tcttcattga tcataacaca aagactacaa cctgggaaga tccacgtttg taaggatcct
     1081 cgagaattca tcgtgactga ctgacgatct gcctcgcgcg tttcggtgat gacggtgaaa
     1141 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     1201 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     1261 cccagtcacg tagcgatagc ggagtgtata attcttgaag acgaaagggc ctcgtgatac
     1321 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     1381 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1441 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1501 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1561 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1621 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1681 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1741 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1801 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1861 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1921 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     1981 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2041 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2101 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2161 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     2221 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     2281 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2341 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2401 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2461 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2521 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2581 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2641 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2701 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2761 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2821 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2881 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2941 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3001 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3061 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3121 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3181 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3241 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3301 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcataaatt
     3361 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
     3421 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
     3481 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
     3541 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
     3601 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
     3661 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
     3721 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
     3781 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
     3841 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
     3901 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
     3961 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
     4021 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
     4081 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
     4141 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
     4201 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
     4261 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
     4321 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     4381 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     4441 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     4501 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     4561 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     4621 taacaatttc acacaggaaa cagctatgac catgattacg gattcactgg ccgtcgtttt
     4681 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     4741 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     4801 gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg gcaccagaag cggtgccgga
     4861 aagctggctg gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct caaactggca
     4921 gatgcacggt tacgatgcgc ccatctacac caacgtaacc tatcccatta cggtcaatcc
     4981 gccgtttgtt cccacggaga atccgacggg ttgttactcg ctcacattta atgttgatga
     5041 aagctggcta caggaaggcc agacgcgaat tatttttgat ggcgttggaa tt
//
