LOCUS       sequence-333883-        5795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses an N-terminal fusion of AMOT PPxY1 to NEDD4L WW3 in 
            bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5795)
  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 5795)
  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: pCA528-PPxY1-GS-WW3e
FEATURES             Location/Qualifiers
     source          1..5795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     921..940
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(960..1037)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1243..1262
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1346..1365
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1346..1364
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1365..1389
                     /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)."
     RBS             1404..1426
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1440..1457
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1461..1751
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             2005..2022
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2075..2093)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2089..2136
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2173..2628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2260..2279)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2470..2491
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2720..3535)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2720..3535)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3443..3462
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3527..3546
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3657..4245
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4146..4165
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4399..4416
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4431..4570
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4496..4515)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4656..4678
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4672..4863)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5672..5795,1..959))
                     /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"
ORIGIN
        1 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
       61 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
      121 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
      181 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
      241 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
      301 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
      361 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
      421 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
      481 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
      541 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
      601 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
      661 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
      721 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
      781 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
      841 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
      901 actgtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
      961 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     1021 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     1081 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     1141 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     1201 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     1261 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     1321 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     1381 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata catatgggtc
     1441 atcaccatca tcaccatggt tcggactcag aagtcaatca agaagctaag ccagaggtca
     1501 agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga tcttcagaga
     1561 tcttcttcaa gatcaaaaag accactcctt taagaaggct gatggaagcg ttcgctaaaa
     1621 gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga attcaagctg
     1681 atcagacccc tgaagatttg gacatggagg ataacgatat tattgaggct cacagagaac
     1741 agattggtgg gatgcagaac aacgaagaac tgccgaccta tgaagaagcg aaagtgggtg
     1801 gcggtggcgg tagcaaagtc acacagagct tcttgccacc cggctgggaa atgaggatag
     1861 cgccaaacgg ccggcccttc ttcattgatc ataacacaaa gactacaacc tgggaagatc
     1921 cacgtttgaa atttccagta catatgcggt caaagtaagg atccgaattc gagctccgtc
     1981 gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac
     2041 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     2101 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     2161 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2221 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2281 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     2341 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     2401 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     2461 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     2521 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     2581 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     2641 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2701 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     2761 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     2821 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     2881 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     2941 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     3001 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     3061 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     3121 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     3181 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     3241 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     3301 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     3361 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     3421 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     3481 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     3541 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     3601 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3661 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3721 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3781 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     3841 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3901 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3961 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4021 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4081 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4141 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4201 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4261 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4321 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4381 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     4441 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     4501 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     4561 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4621 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4681 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     4741 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     4801 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     4861 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     4921 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     4981 tgagggtaaa caactggcgg tattgatgcg gcgggaccag agaaaaatca ctcagggtca
     5041 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     5101 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     5161 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     5221 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     5281 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     5341 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     5401 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     5461 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     5521 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     5581 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     5641 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5701 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5761 gcgccagggt ggtttttctt ttcaccagtg agacg
//
