LOCUS       sequence-203673-        6888 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of WW domain #2 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  JOURNAL   Unpublished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            "Unpublished"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 #2
FEATURES             Location/Qualifiers
     source          1..6888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             138..155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             171..824
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     782..804
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             831..854
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             867..884
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             906..926
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      1083..1130
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2723..3178)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2860..2881)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3072..3091
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3220..3242)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3328..3468
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3383..3402
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3483..3500)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3654..4241)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4412..5203,5204..5272))
                     /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     5035..5054
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5273..5377)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5445..5463
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5486..5563
                     /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             5564..6646
                     /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(5583..5602)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga
      121 ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt atgtccccta
      181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
      841 tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc gcggcccagc
      901 cggccgaaaa cctgtatttt cagggtcgtc gtgcaagcgt ggagctcacc acgccgggtc
      961 tgccgagcgg ttgggaagaa cgtaaagatg cgaaaggccg cacgtattac gtgaaccata
     1021 acaatcgtac cacgacctgg acccgcccga ttatgcagct ggcctaggcg gccgcataat
     1081 aaaccgatac aattaaaggc tccttttgga gccttttttt tggagatttt caacgtgaaa
     1141 aaattattat tcgcaattcc tttagttgtt cctttctatt ctcactccgc tgaaactgtt
     1201 gaaagttgtt tagcaaaacc ccatacagaa aattcattta ctaacgtctg gaaagacgac
     1261 aaaactttag atcgttacgc taactatgag ggctgtctgt ggaatgctac aggcgttgta
     1321 gtttgtactg gtgacgaaac tcagtgtcta gctagagtgg cggtggctct ggttccggtg
     1381 attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa aatgccgatg
     1441 aaaacgcgct acagtctgac gctaaaggca aacttgattc tgtcgctact gattacggtg
     1501 ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat ggtgctactg
     1561 gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt
     1621 taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa tgtcgccctt
     1681 ttgtctttgg cgctggtaaa ccatatgaat tttctattga ttgtgacaaa ataaacttat
     1741 tccgtggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta ttttctacgt
     1801 ttgctaacat actgcgtaat aaggagtctt aatcatgcca gttcttttgg ctagcgccgc
     1861 cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac
     1921 ctgaatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat tggagccaat
     1981 caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat atccatcgcg
     2041 tccgccatct ccagcagccg cacgcggcgc atctcgggca gcgttgggtc ctggccacgg
     2101 gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact
     2161 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg
     2221 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa
     2281 acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg
     2341 ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt
     2401 tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac
     2461 cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc
     2521 attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa
     2581 accgccctta acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc
     2641 aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat
     2701 gagctttacc gcaggatccg cgaaattgta agcgttaata ttttgttaaa attcgcgtta
     2761 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     2821 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     2881 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     2941 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     3001 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     3061 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     3121 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg
     3181 cggatcctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     3241 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     3301 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     3361 agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
     3421 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
     3481 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3541 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3601 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3661 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3721 ccgacaggac tataaagata ccagggtttc cccctggaag ctccctcgtg cgctctcctg
     3781 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3841 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3901 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3961 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4021 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4081 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4141 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4201 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4261 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4321 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4381 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4441 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4501 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4561 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4621 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4681 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg
     4741 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4801 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4861 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4921 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4981 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata
     5041 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5101 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5161 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5221 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5281 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5341 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5401 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     5461 ggccctttcg tcttcaagaa ttcccgacac catcgaatgg tgcaaaacct ttcgcggtat
     5521 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     5581 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     5641 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     5701 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     5761 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     5821 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     5881 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     5941 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     6001 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     6061 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     6121 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     6181 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     6241 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     6301 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
     6361 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     6421 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     6481 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     6541 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     6601 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     6661 atgtaagtta gctcactcat taggcacaat tctcatgttt gacagcttat catcgactgc
     6721 acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag
     6781 gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg gataatgttt
     6841 tttgcgccga catcataacg gttctggcaa atattctgaa atgagctg
//
