LOCUS       sequence-177289-        6035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of E. coli SRAP ortholog YedK.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6035)
  AUTHORS   Kweon SM, Zhu B, Chen Y, Aravind L, Xu SY, Feldman DE
  TITLE     Erasure of Tet-Oxidized 5-Methylcytosine by a SRAP Nuclease.
  JOURNAL   Cell Rep. 2017 Oct 10;21(2):482-494. doi: 
            10.1016/j.celrep.2017.09.055.
  PUBMED    29020633
REFERENCE   2  (bases 1 to 6035)
  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"; doi: 
            "10.1016/j.celrep.2017.09"; journalName: "Cell Rep"; date: 
            "2017-10-10- 10"; volume: "21"; issue: "2"; pages: "482-494"
COMMENT     Sequence Label: pET30a-YedK
FEATURES             Location/Qualifiers
     source          1..6035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     375..394
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     628..645
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    660..799
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(725..744)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     885..907
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(901..1092)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1901..2983)
                     /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     2945..2964
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2984..3061)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3267..3286
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3374..3393
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3374..3392
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3393..3417
                     /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)."
     RBS             3432..3454
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3465..3482
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3492..3509
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3516..3560
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             4233..4259
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4269..4286
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4339..4357)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4353..4400
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4437..4892
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4524..4543)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4734..4755
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4984..5799)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4984..5799)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5707..5726
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5791..5810
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(5921..6035,1..474)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
       61 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
      121 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
      181 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
      241 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
      301 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
      361 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
      421 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
      481 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
      541 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
      601 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
      661 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
      721 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
      781 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
      841 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
      901 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
      961 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     1021 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     1081 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     1141 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     1201 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     1261 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     1321 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     1381 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     1441 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     1501 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     1561 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     1621 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     1681 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     1741 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     1801 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     1861 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     1921 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     1981 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     2041 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     2101 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     2161 cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc
     2221 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     2281 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     2341 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     2401 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     2461 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     2521 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     2581 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     2641 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     2701 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     2761 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     2821 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     2881 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     2941 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     3001 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     3061 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     3121 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     3181 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     3241 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     3301 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatcgatctc
     3361 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     3421 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgcaccat catcatcatc
     3481 attcttctgg tctggtgcca cgcggttctg gtatgaaaga aaccgctgct gctaaattcg
     3541 aacgccagca catggacagc ccagatctgt gtggacgctt tgcccaatcc caaacgcgtg
     3601 aagattacct tgcgcttctc gcggaagata ttgaacgcga tattccctat gatcccgaac
     3661 ccattggcag atacaacgtc gcgccgggaa ccaaagtcct actgctcagt gaacgtgatg
     3721 aacaccttca tctggatccg gttttctggg gatatgctcc cggatggtgg gataaaccgc
     3781 cgctgattaa cgcccgcgta gaaactgcgg caaccagtcg tatgtttaaa ccgctctggc
     3841 aacatggtcg ggcaatctgt tttgccgatg gctggtttga gtggaaaaaa gaaggcgaca
     3901 aaaaacagcc tttttttatc tatcgtgctg atggacaacc tatttttatg gccgcgatag
     3961 gtagcacacc atttgagcgt ggtgacgaag ccgaaggatt tttgatcgtc actgctgcgg
     4021 cagatcaagg tctggtagat attcatgacc gccgcccact ggtactgtcg ccagaagctg
     4081 cgcgggaatg gatgcggcaa gagattagcg gtaaagaagc ctcagaaatc gcggccagtg
     4141 gctgtgttcc agcaaaccag ttctcctggc acccggtatc gcgcgcggtg ggtaatgtta
     4201 aaaaccaggg cgcggagtta attcaacctg tttacccata cgatgttcca gattacgctg
     4261 gactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     4321 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     4381 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     4441 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4501 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4561 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4621 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     4681 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4741 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     4801 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4861 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     4921 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     4981 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     5041 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     5101 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     5161 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     5221 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     5281 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     5341 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     5401 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     5461 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     5521 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     5581 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     5641 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     5701 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     5761 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     5821 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5881 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5941 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6001 atcaagagct accaactctt tttccgaagg taact
//
