LOCUS       sequence-398149-        5639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal GST fusion of Af1521(WT) with a TEV protease site located
            between the GST tag and Af1521 and a myc tag on the C terminus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5639)
  AUTHORS   Al-Rahahleh RQ, Roos WP, Saville KM, Andrews JF, Wu Z, Koczor CA, 
            Prakash A, Sobol RW
  TITLE     Overexpression of the WWE domain of RNF146 modulates 
            poly-(ADP)-ribose dynamics at sites of DNA damage.
  JOURNAL   DNA Repair (Amst). 2025 Jun;150:103845. doi: 
            10.1016/j.dnarep.2025.103845. Epub 2025 May 21.
  PUBMED    40403420
REFERENCE   2  (bases 1 to 5639)
  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: "DNA Repair 
            (Amst)."; date: "2025-06"; volume: "150:103845. doi"; pages: " 
            10.1016/j.dnarep.2025.103845. Epub 2025 May 21"
COMMENT     Sequence Label: pGEX-4T-3-GST-Tev-Af1521-myc
FEATURES             Location/Qualifiers
     source          1..5639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(181..186,187..202,203..209)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    217..233
                     /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             256..909
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     867..889
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             916..933
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             952..972
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     RBS             1473..1481
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             1594..1623
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1687..1709)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1854..1872)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1940..2044
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2045..2113,2114..2905)
                     /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(2263..2282)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3076..3664
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3565..3584
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3818..3835
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3908..3985
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3986..5068
                     /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(4005..4024)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5117..5122,5123..5140,5141..5147)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5155..5171
                     /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     5179..5195
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5179..5195
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5191..5639,1..79)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5211..5228)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5211..5227)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5220..5242)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cttatcgact gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt
       61 atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc actcccgttc
      121 tggataatgt tttttgcgcc gacatcataa cggttctggc aaatattctg aaatgagctg
      181 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
      241 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      301 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      361 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      421 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      481 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      541 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      601 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      661 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      721 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      781 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      841 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      901 cctccaaaat cggatctggt tccgcgtgga tccccgaatt cccgggtcga cgagaattta
      961 tattttcaag gagaagtact attcgaagcg aaggtgggtg acatcactct gaagttagca
     1021 caaggtgaca tcacgcagta tccggcgaag gctattgtta acgcagcgaa caagcgtctc
     1081 gagcacggcg gcggcgtagc gtatgcaatc gccaaggcgt gcgcgggtga tgctggcctg
     1141 tacaccgaaa tttccaaaaa ggctatgcgt gaacaatttg gccgtgatta catcgaccac
     1201 ggtgaagtgg tcgttacccc agccatgaat ctggaggagc gcggtatcaa atacgtgttt
     1261 cataccgttg gtccgatttg tagcggcatg tggagcgaag agctgaagga gaagctgtac
     1321 aaagccttcc tgggtccgct ggaaaaggcg gaggagatgg gtgtcgaatc cattgcgttt
     1381 ccggcggtta gcgcgggcat ctacggctgc gacctggaga aagtggtgga aacctttttg
     1441 gaagcagtta aaaacttcaa aggttcggcc gttaaggagg tcgctttggt tatctatgat
     1501 cgtaaaagcg cggaagtggc tcttaaagtg ttcgagcgca gcttgagcgg gggtggtggc
     1561 tctggtggag gcggctctgg tggtggcggt tccgagcaga aactgattag cgaggaagat
     1621 ttgtaagcgg ccgcatcgtg actgactgac gatctgcctc gcgcgtttcg gtgatgacgg
     1681 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     1741 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     1801 catgacccag tcacgtagcg atagcggagt gtataattct tgaagacgaa agggcctcgt
     1861 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     1921 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1981 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2041 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     2101 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     2161 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     2221 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     2281 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     2341 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     2401 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     2461 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     2521 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     2581 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     2641 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     2701 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     2761 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     2821 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     2881 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     2941 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     3001 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3061 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3121 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3181 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     3241 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3301 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3361 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3421 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3481 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3541 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3601 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3661 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3721 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3781 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3841 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3901 aaattccgac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga
     3961 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     4021 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     4081 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     4141 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     4201 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     4261 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     4321 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     4381 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     4441 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     4501 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     4561 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     4621 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     4681 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     4741 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
     4801 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     4861 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     4921 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     4981 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     5041 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     5101 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     5161 gcggataaca atttcacaca ggaaacagct atgaccatga ttacggattc actggccgtc
     5221 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     5281 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     5341 cagttgcgca gcctgaatgg cgaatggcgc tttgcctggt ttccggcacc agaagcggtg
     5401 ccggaaagct ggctggagtg cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac
     5461 tggcagatgc acggttacga tgcgcccatc tacaccaacg tgacctatcc cattacggtc
     5521 aatccgccgt ttgttcccac ggagaatccg acgggttgtt actcgctcac atttaatgtt
     5581 gatgaaagct ggctacagga aggccagacg cgaattattt ttgatggcgt tggaattag
//
