LOCUS       sequence-202156-        6008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses specific cyclic-ADP-ribose (cADPR) phosphohydrolase in 
            Escherichia coli as a GST fusion protein that can be cut with 
            PreScission(TM) protease to yield active enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6008)
  AUTHORS   Ribeiro JM, Canales J, Cabezas A, Rodrigues JR, Pinto RM, 
            Lopez-Villamizar I, Costas MJ, Cameselle JC
  TITLE     Specific cyclic ADP-ribose phosphohydrolase obtained by mutagenic 
            engineering of Mn(2+)-dependent ADP-ribose/CDP-alcohol 
            diphosphatase.
  JOURNAL   Sci Rep. 2018 Jan 18;8(1):1036. doi: 10.1038/s41598-017-18393-9.
  PUBMED    29348648
REFERENCE   2  (bases 1 to 6008)
  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: "Sci Rep."; 
            date: "2018-01-18"; volume: "8(1):1036. doi"; pages: " 
            10.1038/s41598-017-18393-9"
COMMENT     Sequence Label: pGEX-6P-3-F37A+L196F+V252A+C253G-hADPRM
FEATURES             Location/Qualifiers
     source          1..6008
                     /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..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2058..2080)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2225..2243)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2311..2415
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2416..2484,2485..3276)
                     /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(2634..2653)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3447..4035
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3936..3955
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4189..4206
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4279..4356
                     /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             4357..5439
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4376..4395)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5452..5473
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5488..5493,5494..5511,5512..5518)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5526..5542
                     /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     5550..5566
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5550..5566
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5562..6008,1..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(5582..5599)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5582..5598)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5591..5613)
                     /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 gaagttctgt tccaggggcc cctgggatcc ccgaattccc
      961 gggtcgacat ggatgataaa cccaaccctg aagccctaag tgacagttca gagcgtcttt
     1021 tctcctttgg cgtcatcgca gatgttcaat ttgcagactt agaagatggc tttaatgcac
     1081 aaggaaccag gcggcgatac tacagacata gtcttcttca cttacagggt gccattgaag
     1141 actggaataa tgaaagcagc atgccctgtt gtgtccttca gcttggagat atcatcgatg
     1201 gatataatgc acagtataat gcatccaaaa agtccctaga acttgttatg gacatgttca
     1261 agaggcttaa agttccagtt catcatacat ggggaaacca tgaattctat aacttcagta
     1321 gagagtattt aacacactct aaacttaaca ctaagtttct agaagatcag attgtacatc
     1381 atcctgagac catgccttca gaagattatt atgcttatca ttttgtacca ttccctaaat
     1441 tccggttcat tttacttgat gcatatgact tgagtgtctt gggcgtggat cagtcttctc
     1501 caaaatacga gcagtgtatg aagatattga gggagcacaa tccaaatacg gaattcaata
     1561 gccctcaagg actttctgag ccccagtttg tccagtttaa tggaggattc agccaagaac
     1621 agctaaactg gttgaatgaa gtgctaacat tctctgacac aaaccaagaa aaggtggtga
     1681 ttgtgagcca tcttcccatt tacccggacg cgtctgacaa tgctggcctg gcctggaact
     1741 acagagatgc cctggcagtc atttggtctc atgagtgtgt ggtgtgtttc tttgctggtc
     1801 acacccatga tggtggctac tctgaggatc cttttggtgt ataccacgtc aacctagaag
     1861 gagttattga aacagctcca gacagccaag cctttggcac agttcatgtc tatcctgaca
     1921 aaatgatgtt gaaagggaga ggcagagttc cagatagaat tatgaattac aagaaagaaa
     1981 gagcattcca ttgttaggcg gccgcatcgt gactgactga cgatctgcct cgcgcgtttc
     2041 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     2101 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     2161 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtataattc ttgaagacga
     2221 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     2281 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2341 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2401 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2461 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2521 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2581 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2641 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     2701 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2761 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2821 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2881 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2941 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3001 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3061 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3121 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3181 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3241 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3301 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3361 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3421 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3481 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3541 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3601 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3661 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3721 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3781 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3841 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3901 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3961 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4021 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4081 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4141 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4201 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4261 tcacaccgca taaattccga caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga
     4321 tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat
     4381 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc
     4441 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt
     4501 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc
     4561 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat
     4621 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa
     4681 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg
     4741 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt
     4801 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga
     4861 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca
     4921 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
     4981 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
     5041 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag
     5101 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc
     5161 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gtcaaccacc
     5221 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
     5281 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
     5341 accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     5401 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     5461 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     5521 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacggatt
     5581 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     5641 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     5701 gcccttccca acagttgcgc agcctgaatg gcgaatggcg ctttgcctgg tttccggcac
     5761 cagaagcggt gccggaaagc tggctggagt gcgatcttcc tgaggccgat actgtcgtcg
     5821 tcccctcaaa ctggcagatg cacggttacg atgcgcccat ctacaccaac gtaacctatc
     5881 ccattacggt caatccgccg tttgttccca cggagaatcc gacgggttgt tactcgctca
     5941 catttaatgt tgatgaaagc tggctacagg aaggccagac gcgaattatt tttgatggcg
     6001 ttggaatt
//
