LOCUS       sequence-259243-        5662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low level expression of E.coli G.stearothermophillus MutY chimera 
            protein with amino acid changes F307A and S308A in Gs MutY..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5662)
  AUTHORS   Russelburg LP, O'Shea Murray VL, Demir M, Knutsen KR, Sehgal SL, Cao
            S, David SS, Horvath MP
  TITLE     Structural Basis for Finding OG Lesions and Avoiding Undamaged G by 
            the DNA Glycosylase MutY.
  JOURNAL   ACS Chem Biol. 2019 Dec 27. doi: 10.1021/acschembio.9b00639.
  PUBMED    31829624
REFERENCE   2  (bases 1 to 5662)
  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: "ACS Chem 
            Biol. 2019 Dec 27. doi: 10.1021/acschembio.9b00639."
COMMENT     Sequence Label: EcNGsC-F307A-S308A-pKK223
FEATURES             Location/Qualifiers
     source          1..5662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1153..1170)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1323..1409
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1501..1528
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1547..1638
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1639..1707,1708..2499)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1857..1876)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2670..3258
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3159..3178
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3412..3429
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3444..3584
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3510..3529)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3670..3692
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3686..3877)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5335..5354
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(5601..5606,5607..5622,5623..5629)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5637..5653
                     /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     join(5642..5662,1..2)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 ggaaacagaa ttcggtgacc atgcaagcgt cgcaattttc agcccaggtt ctggactggt
       61 acgataaata cgggcgaaaa actctgccct ggcaaattga caagacgccc tacaaagtat
      121 ggctctcaga agtgatgttg caacaaactc aggttgcgac cgttatcccc tattttgaac
      181 gctttatggc gcgcttcccg acggtgaccg atctcgccaa tgcgccgctc gacgaagttc
      241 tccacttgtg gaccgggctt ggctattacg cccgcgcgcg caatctgcat aaagcggcac
      301 aacaagtggc gaccttacac ggcggtaaat tcccggaaac ctttgaggaa gttgcagcac
      361 tgccgggcgt cgggcgttcc accgcaggcg cgattctctc gctttctctg ggtaagcact
      421 ttccgattct cgacggtaac gtcaaacgcg tgctggcgcg ctgctatgct gtaagcggct
      481 ggcctgggaa aaaagaggtc gagaataaat tatggagttt gagcgagcag gtgacgcccg
      541 cggttggcgt ggaacggttt aatcaggcga tgatggattt gggtgcgatg atttgtacgc
      601 gctcgaaacc gaaatgttcg ctctgtccgc tacaaaacgg atgtattgcc gccgccaaca
      661 atagctgggc gctttatccg ggcaaaaaac cgaaaaaaac agcggtcaaa caagtgccgc
      721 tcgcagtagc ggtgctggcc gatgatgaag gccgcgttct cattcgcaag cgcgacagca
      781 ccggtttgct tgcgaactta tgggagttcc ccagctgcga aacggacggc gcagatggaa
      841 aagagaagct cgagcaaatg gttggggagc aatacggttt gcaagtggag cttacagaac
      901 cgatcgtttc gtttgagcat gcggctgccc atttagtctg gcagttgacc gtgtttcccg
      961 gccgtttggt tcatggcgga ccggtcgagg agccttaccg gcttgcgccg gaagacgagt
     1021 tgaaggcgta cgcctttccg gtgtcccatc aacgtgtttg gcgcgaatat aaagaatggg
     1081 cgagcggagt acgcccgccc ccatgactgc agccaagctt ggctgttttg gcggatgaga
     1141 gaagattttc agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa
     1201 tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa
     1261 acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc
     1321 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     1381 cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc
     1441 aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc
     1501 agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttttg tttatttttc
     1561 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     1621 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     1681 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     1741 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     1801 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     1861 tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac
     1921 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     1981 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     2041 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     2101 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     2161 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     2221 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     2281 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     2341 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     2401 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     2461 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     2521 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     2581 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2641 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2701 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2761 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     2821 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2881 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2941 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3001 tgcacacagc ccagcttgga gcgaacgaac tacaccgaac tgagatacct acagcgtgag
     3061 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3121 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3181 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3241 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3301 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3361 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3421 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3481 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3541 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     3601 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3661 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3721 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     3781 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     3841 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     3901 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     3961 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     4021 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     4081 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     4141 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     4201 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     4261 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     4321 ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc
     4381 ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca
     4441 cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt
     4501 gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg
     4561 gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc
     4621 cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag
     4681 agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat
     4741 ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc
     4801 catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc
     4861 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     4921 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     4981 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5041 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5101 ggagctgact gggttgaagg ctctcaaggg catcggtcga cgctctccct tatgcgactc
     5161 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     5221 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     5281 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     5341 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     5401 tccggcgtag aggatccggg cttatcgact gcacggtgca ccaatgcttc tggcgtcagg
     5461 cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg
     5521 ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa cggttctggc
     5581 aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt gtggaattgt
     5641 gagcggataa caatttcaca ca
//
