LOCUS       sequence-398155-        5639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal GST fusion of Af1521(K35E/Y145R) 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(K35E/Y145R)-myc
FEATURES             Location/Qualifiers
     source          1..5639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(182..187,188..203,204..210)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    218..234
                     /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             257..910
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     868..890
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             917..934
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             953..973
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1595..1624
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1688..1710)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1855..1873)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1941..2045
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2046..2114,2115..2906)
                     /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(2264..2283)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3077..3665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3566..3585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3819..3836
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3909..3986
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3987..5069
                     /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(4006..4025)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5118..5123,5124..5141,5142..5148)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5156..5172
                     /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     5180..5196
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5180..5196
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5192..5639,1..80)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5212..5229)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5212..5228)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5221..5243)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gcttatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
       61 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
      121 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
      181 gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac
      241 acaggaaaca gtattcatgt cccctatact aggttattgg aaaattaagg gccttgtgca
      301 acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg
      361 cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct
      421 tccttattat attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat
      481 agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct
      541 tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt
      601 tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga
      661 tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt
      721 gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa
      781 attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc
      841 cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca
      901 tcctccaaaa tcggatctgg ttccgcgtgg atccccgaat tcccgggtcg acgagaattt
      961 atattttcaa ggagaagtac tattcgaagc aaaggtgggt gacatcaccc tgaagctggc
     1021 gcagggtgac atcacccagt atccggcgaa agcgattgtt aacgctgcga acgaacgcct
     1081 ggagcacggt ggcggcgttg cctatgcgat cgccaaggcg tgtgcaggtg acgcaggtct
     1141 gtacaccgaa atttccaaaa aagctatgcg tgaacaattt ggtcgtgatt atatcgacca
     1201 cggcgaagtg gtggttactc cggctatgaa tctggaagaa agaggtatca aatacgtgtt
     1261 tcataccgtc ggtccgattt gcagcggcat gtggtcggaa gagttgaaag aaaagctcta
     1321 caaagccttc ctgggtccac tggagaaggc ggaagagatg ggtgtcgaaa gcattgcatt
     1381 tccggcggtt agcgctggca tccgtggttg cgatctggag aaggttgtcg agacgttttt
     1441 agaggcggtt aagaacttca aaggtagtgc tgtgaaggag gtggcgttgg ttatttacga
     1501 tcgtaaaagc gcggaagtag ccttgaaggt gttcgagcgc tctctgagcg gtggcggtgg
     1561 ctctggtggc ggcggttccg gcgggggcgg ctccgagcaa aaactgatca gcgaggagga
     1621 tctttaagcg gccgcatcgt gactgactga cgatctgcct cgcgcgtttc ggtgatgacg
     1681 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     1741 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     1801 ccatgaccca gtcacgtagc gatagcggag tgtataattc ttgaagacga aagggcctcg
     1861 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     1921 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1981 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2041 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     2101 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     2161 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     2221 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     2281 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2341 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2401 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     2461 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     2521 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     2581 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     2641 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     2701 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     2761 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     2821 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     2881 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     2941 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     3001 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3061 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3781 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3841 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3901 taaattccga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     3961 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
     4021 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     4081 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     4141 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     4201 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     4261 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     4321 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     4381 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     4441 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
     4501 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     4561 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
     4621 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
     4681 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     4741 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg
     4801 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     4861 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     4921 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     4981 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     5041 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact
     5101 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     5161 agcggataac aatttcacac aggaaacagc tatgaccatg attacggatt cactggccgt
     5221 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     5281 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     5341 acagttgcgc agcctgaatg gcgaatggcg ctttgcctgg tttccggcac cagaagcggt
     5401 gccggaaagc tggctggagt gcgatcttcc tgaggccgat actgtcgtcg tcccctcaaa
     5461 ctggcagatg cacggttacg atgcgcccat ctacaccaac gtgacctatc ccattacggt
     5521 caatccgccg tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt
     5581 tgatgaaagc tggctacagg aaggccagac gcgaattatt tttgatggcg ttggaatta
//
