LOCUS       sequence-217281-        7254 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of VCP_AAA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7254)
  AUTHORS   Teyra J, Kelil A, Jain S, Helmy M, Jajodia R, Hooda Y, Gu J, D'Cruz 
            AA, Nicholson SE, Min J, Sudol M, Kim PM, Bader GD, Sidhu SS
  TITLE     Large-scale survey and database of high affinity ligands for peptide
            recognition modules.
  JOURNAL   Mol Syst Biol. 2020 Dec;16(12):e9310. doi: 10.15252/msb.20199310.
  PUBMED    33438817
REFERENCE   2  (bases 1 to 7254)
  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: "Mol Syst 
            Biol."; date: "2020-12"; volume: "16(12):e9310. doi"; pages: " 
            10.15252/msb.20199310"
COMMENT     Sequence Label: pHH0103_VCP_AAA
FEATURES             Location/Qualifiers
     source          1..7254
                     /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"
     terminator      1449..1496
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3089..3544)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3226..3247)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3438..3457
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3586..3608)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3694..3834
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3749..3768
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3849..3866)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4020..4607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4778..5569,5570..5638))
                     /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     5401..5420
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5639..5743)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5811..5829
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5852..5929
                     /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             5930..7012
                     /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(5949..5968)
                     /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 cggcccaggt tacatgggaa gatattggtg gtctggaaga tgttaaacgt gaactgcaag
      961 aactggttca gtatccggtt gaacatccgg ataaatttct gaagtttggt atgaccccga
     1021 gcaaaggtgt tctgttttat ggtccgcctg gttgtggtaa aaccctgctg gcaaaagcaa
     1081 ttgcaaatga atgtcaggcc aacttcatta gcattaaagg tccggaactg ctgaccatgt
     1141 ggtttggtga aagcgaagca aatgtgcgcg aaatttttga taaagcacgt caggcagcac
     1201 cgtgtgtgct gttttttgat gaactggata gcattgcaaa agcccgtggt ggtaacattg
     1261 gtgatggtgg tggtgcagca gatcgtgtta ttaatcagat tctgaccgaa atggatggta
     1321 tgagcaccaa aaaaaacgtg tttattatcg gtgcaaccaa ccgtccggat attattgatc
     1381 cggcaattct gcgtccgggt cgtctggatc aactgattta tatcccgctg cctgcggccg
     1441 cataataaac cgatacaatt aaaggctcct tttggagcct tttttttgga gattttcaac
     1501 gtgaaaaaat tattattcgc aattccttta gttgttcctt tctattctca ctccgctgaa
     1561 actgttgaaa gttgtttagc aaaaccccat acagaaaatt catttactaa cgtctggaaa
     1621 gacgacaaaa ctttagatcg ttacgctaac tatgagggct gtctgtggaa tgctacaggc
     1681 gttgtagttt gtactggtga cgaaactcag tgtctagcta gagtggcggt ggctctggtt
     1741 ccggtgattt tgattatgaa aagatggcaa acgctaataa gggggctatg accgaaaatg
     1801 ccgatgaaaa cgcgctacag tctgacgcta aaggcaaact tgattctgtc gctactgatt
     1861 acggtgctgc tatcgatggt ttcattggtg acgtttccgg ccttgctaat ggtaatggtg
     1921 ctactggtga ttttgctggc tctaattccc aaatggctca agtcggtgac ggtgataatt
     1981 cacctttaat gaataatttc cgtcaatatt taccttccct ccctcaatcg gttgaatgtc
     2041 gcccttttgt ctttggcgct ggtaaaccat atgaattttc tattgattgt gacaaaataa
     2101 acttattccg tggtgtcttt gcgtttcttt tatatgttgc cacctttatg tatgtatttt
     2161 ctacgtttgc taacatactg cgtaataagg agtcttaatc atgccagttc ttttggctag
     2221 cgccgcccta taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac
     2281 ctcgacctga atggaagccg gcggcacctc gctaacggat tcaccactcc aagaattgga
     2341 gccaatcaat tcttgcggag aactgtgaat gcgcaaacca acccttggca gaacatatcc
     2401 atcgcgtccg ccatctccag cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg
     2461 ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc
     2521 cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc
     2581 aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt
     2641 ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc
     2701 tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga
     2761 gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc
     2821 agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc
     2881 ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag
     2941 gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag
     3001 aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac
     3061 gctgatgagc tttaccgcag gatccgcgaa attgtaagcg ttaatatttt gttaaaattc
     3121 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     3181 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     3241 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     3301 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     3361 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     3421 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     3481 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     3541 gcgtcgcgga tcctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     3601 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     3661 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga
     3721 tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
     3781 catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
     3841 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3901 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3961 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4021 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4081 cgaaacccga caggactata aagataccag ggtttccccc tggaagctcc ctcgtgcgct
     4141 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4201 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4261 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4321 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4381 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4441 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4501 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4561 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4621 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4681 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4741 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4801 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4861 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4921 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4981 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5041 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca
     5101 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5161 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5221 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5281 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5341 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg
     5401 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5461 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5521 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5581 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5641 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5701 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5761 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     5821 tcacgaggcc ctttcgtctt caagaattcc cgacaccatc gaatggtgca aaacctttcg
     5881 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
     5941 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     6001 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     6061 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     6121 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     6181 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     6241 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     6301 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     6361 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     6421 agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     6481 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     6541 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     6601 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     6661 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     6721 tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc
     6781 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     6841 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt
     6901 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     6961 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     7021 caattaatgt aagttagctc actcattagg cacaattctc atgtttgaca gcttatcatc
     7081 gactgcacgg tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct
     7141 gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata
     7201 atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga gctg
//
