LOCUS       sequence-204806-        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(160..165,166..183,184..190)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    198..214
                     /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     222..238
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     222..238
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             234..761
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(254..271)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(254..270)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(263..285)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(863..868,869..884,885..891)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    899..915
                     /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             938..1591
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1549..1571
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1598..1621
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2738..2760)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2905..2923)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2991..3095
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3096..3164,3165..3956)
                     /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(3314..3333)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4127..4715
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4616..4635
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4869..4886
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4959..5036
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5037..6008,1..111)
                     /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(5056..5075)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 aaaagaaaaa ccacgctagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
       61 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
      121 aattaatgta agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
      181 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
      241 tgattacgga ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
      301 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
      361 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgctttgcct
      421 ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt cctgaggccg
      481 atactgtcgt cgtcccctca aactggcaga tgcacggtta cgatgcgccc atctacacca
      541 acgtgaccta tcccattacg gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt
      601 gttactcgct cacatttaat gttgatgaaa gctggctaca ggaaggccag acgcgaatta
      661 tttttgatgg cgttggaatt agcttatcga ctgcacggtg caccaatgct tctggcgtca
      721 ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt
      781 cgctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg
      841 gcaaatattc tgaaatgagc tgttgacaat taatcatcgg ctcgtataat gtgtggaatt
      901 gtgagcggat aacaatttca cacaggaaac agtattcatg tcccctatac taggttattg
      961 gaaaattaag ggccttgtgc aacccactcg acttcttttg gaatatcttg aagaaaaata
     1021 tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt
     1081 gggtttggag tttcccaatc ttccttatta tattgatggt gatgttaaat taacacagtc
     1141 tatggccatc atacgttata tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga
     1201 gcgtgcagag atttcaatgc ttgaaggagc ggttttggat attagatacg gtgtttcgag
     1261 aattgcatat agtaaagact ttgaaactct caaagttgat tttcttagca agctacctga
     1321 aatgctgaaa atgttcgaag atcgtttatg tcataaaaca tatttaaatg gtgatcatgt
     1381 aacccatcct gacttcatgt tgtatgacgc tcttgatgtt gttttataca tggacccaat
     1441 gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa cgtattgaag ctatcccaca
     1501 aattgataag tacttgaaat ccagcaagta tatagcatgg cctttgcagg gctggcaagc
     1561 cacgtttggt ggtggcgacc atcctccaaa atcggatctg gaagttctgt tccaggggcc
     1621 cctgggatcc ccgaattccc gggtcgacat ggatgataaa cccaaccctg aagccctaag
     1681 tgacagttca gagcgtcttt tctcctttgg cgtcatcgca gatgttcaat ttgcagactt
     1741 agaagatggc tttaatgcac aaggaaccag gcggcgatac tacagacata gtcttcttca
     1801 cttacagggt gccattgaag actggaataa tgaaagcagc atgccctgtt gtgtccttca
     1861 gcttggagat atcatcgatg gatataatgc acagtataat gcatccaaaa agtccctaga
     1921 acttgttatg gacatgttca agaggcttaa agttccagtt catcatacat ggggaaacca
     1981 tgaattctat aacttcagta gagagtattt aacacactct aaacttaaca ctaagtttct
     2041 agaagatcag attgtacatc atcctgagac catgccttca gaagattatt atgcttatca
     2101 ttttgtacca ttccctaaat tccggttcat tttacttgat gcatatgact tgagtgtctt
     2161 gggcgtggat cagtcttctc caaaatacga gcagtgtatg aagatattga gggagcacaa
     2221 tccaaatacg gaattcaata gccctcaagg actttctgag ccccagtttg tccagtttaa
     2281 tggaggattc agccaagaac agctaaactg gttgaatgaa gtgctaacat tctctgacac
     2341 aaaccaagaa aaggtggtga ttgtgagcca tcttcccatt tacccggacg cgtctgacaa
     2401 tgctggcctg gcctggaact acagagatgc cctggcagtc atttggtctc atgagtgtgt
     2461 ggtgtgtttc tttgctggtc acacccatga tggtggctac tctgaggatc cttttggtgt
     2521 ataccacgtc aacctagaag gagttattga aacagctcca gacagccaag cctttggcac
     2581 agttcatgtc tatcctgaca aaatgatgtt gaaagggaga ggcagagttc cagatagaat
     2641 tatgaattac aagaaagaaa gagcattcca ttgttaggcg gccgcatcgt gactgactga
     2701 cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     2761 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     2821 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     2881 tgtataattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     2941 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3001 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3061 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3121 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3181 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3241 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3301 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     3361 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3421 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3481 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3541 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3601 aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
     3661 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3721 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3781 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     3841 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     3901 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     3961 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4021 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4081 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4141 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4201 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4261 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4321 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4381 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4441 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4501 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4561 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4621 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4681 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4741 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4801 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4861 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     4921 ttacgcatct gtgcggtatt tcacaccgca taaattccga caccatcgaa tggcgcaaaa
     4981 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     5041 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     5101 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     5161 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     5221 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccatcgcaa attgtcgcgg
     5281 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     5341 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     5401 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     5461 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     5521 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     5581 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     5641 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     5701 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     5761 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     5821 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     5881 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     5941 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     6001 cactggtg
//
