LOCUS       sequence-202575-        6888 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of WW domain #3 from NEDD4L.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6888)
  TITLE     Sachdev Sidhu WW domains
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6888)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pHH0103 NEDD4L WW domain #3
FEATURES             Location/Qualifiers
     source          1..6888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..654
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     612..634
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             661..684
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             697..714
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             736..756
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      913..960
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2553..3008)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2690..2711)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2902..2921
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3050..3072)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3158..3298
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3213..3232
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3313..3330)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3484..4071)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4242..5033,5034..5102))
                     /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     4865..4884
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5103..5207)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5275..5293
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5316..5393
                     /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             5394..6476
                     /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(5413..5432)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6719..6724,6725..6740,6741..6747)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    6755..6771
                     /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     6760..6782
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             6856..6873
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt
       61 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa
      121 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat
      181 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac
      241 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg
      301 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt
      361 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa
      421 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat
      481 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa
      541 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca
      601 tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat
      661 ctggaagttc tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc
      721 gcggcccagc cggccgaaaa cctgtatttt cagggtcgtc gtgcaagcgt ggagctcacg
      781 cagagctttc tgccgccggg ctgggaaatg cgtattgcgc cgaacggtcg cccgtttttc
      841 atcgatcata ataccaaaac cacgacctgg gaagatccgc gtctgaaatt cccgtaggcg
      901 gccgcataat aaaccgatac aattaaaggc tccttttgga gccttttttt tggagatttt
      961 caacgtgaaa aaattattat tcgcaattcc tttagttgtt cctttctatt ctcactccgc
     1021 tgaaactgtt gaaagttgtt tagcaaaacc ccatacagaa aattcattta ctaacgtctg
     1081 gaaagacgac aaaactttag atcgttacgc taactatgag ggctgtctgt ggaatgctac
     1141 aggcgttgta gtttgtactg gtgacgaaac tcagtgtcta gctagagtgg cggtggctct
     1201 ggttccggtg attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa
     1261 aatgccgatg aaaacgcgct acagtctgac gctaaaggca aacttgattc tgtcgctact
     1321 gattacggtg ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat
     1381 ggtgctactg gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat
     1441 aattcacctt taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa
     1501 tgtcgccctt ttgtctttgg cgctggtaaa ccatatgaat tttctattga ttgtgacaaa
     1561 ataaacttat tccgtggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta
     1621 ttttctacgt ttgctaacat actgcgtaat aaggagtctt aatcatgcca gttcttttgg
     1681 ctagcgccgc cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg
     1741 ccacctcgac ctgaatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat
     1801 tggagccaat caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat
     1861 atccatcgcg tccgccatct ccagcagccg cacgcggcgc atctcgggca gcgttgggtc
     1921 ctggccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg
     1981 ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg
     2041 ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta
     2101 aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg
     2161 atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc
     2221 ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg
     2281 ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt
     2341 catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa
     2401 acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat taacgcttct
     2461 ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga
     2521 ccacgctgat gagctttacc gcaggatccg cgaaattgta agcgttaata ttttgttaaa
     2581 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     2641 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     2701 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     2761 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     2821 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     2881 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     2941 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     3001 gggcgcgtcg cggatcctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     3061 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     3121 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     3181 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt
     3241 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     3301 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3361 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3421 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3481 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3541 gtggcgaaac ccgacaggac tataaagata ccagggtttc cccctggaag ctccctcgtg
     3601 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3661 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3721 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3781 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3841 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3901 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     3961 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4021 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4081 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4141 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4201 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4261 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4321 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4381 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4441 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4501 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca
     4561 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4621 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4681 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4741 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4801 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca
     4861 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4921 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4981 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5041 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5101 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5161 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5221 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     5281 cgtatcacga ggccctttcg tcttcaagaa ttcccgacac catcgaatgg tgcaaaacct
     5341 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
     5401 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
     5461 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     5521 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
     5581 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
     5641 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     5701 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     5761 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     5821 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     5881 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     5941 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     6001 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
     6061 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
     6121 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
     6181 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
     6241 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     6301 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     6361 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
     6421 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     6481 aacgcaatta atgtaagtta gctcactcat taggcacaat tctcatgttt gacagcttat
     6541 catcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat
     6601 ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg
     6661 gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt
     6721 gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca
     6781 ggaaacagcc agtccgttta ggtgttttca cgagcacttc accaacaagg accatagatt
     6841 atgaaaatcg aagaacacca tcaccatcac cattccagcg gtaagctt
//
