LOCUS       40924_10161        8104 bp DNA     circular SYN 13-MAY-2021
DEFINITION  To introduce MS2 coat protein fused Tet1-CD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8104)
  AUTHORS   Xu X, Tao Y, Gao X, Zhang L, Li X, Zou W, Ruan K, Wang F, Xu GL, Hu 
            R
  TITLE     A CRISPR-based approach for targeted DNA demethylation.
  JOURNAL   Cell Discov. 2016 May 3;2:16009. doi: 10.1038/celldisc.2016.9. 
            eCollection 2016.
  PUBMED    27462456
REFERENCE   2  (bases 1 to 8104)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8104)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell
            Discov."; date: "2016-05-3"; pages: "
            10.1038/celldisc.2016.9. eCollection 2016"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             926..1312
                     /codon_start=1
                     /label=MS2-N55K
                     /note="bacteriophage MS2 coat protein"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             1364..1384
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             3461..3484
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     polyA_signal    3531..3755
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3801..4229
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        4243..4573
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             4622..5644
                     /codon_start=1
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    5777..5910
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5947..5963)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5971..5987)
                     /label=lac operator
                     /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)."
     promoter        complement(5995..6025)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6040..6061)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(6178..6195)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6349..6937)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7111..7968)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7969..8073)
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccatggcttc taactttact cagttcgttc tcgtcgacaa
      961 tggcggaact ggcgacgtga ctgtcgcccc aagcaacttc gctaacgggg tcgctgaatg
     1021 gatcagctct aactcgcgtt cacaggctta caaagtaacc tgtagcgttc gtcagagctc
     1081 tgcgcagaag cgcaaataca ccatcaaagt cgaggtgcct aaagtggcaa cccagactgt
     1141 tggtggtgta gagcttcctg tagccgcatg gcgttcgtac ttaaatatgg aactaaccat
     1201 tccaattttc gctacgaatt ccgactgcga gcttattgtt aaggcaatgc aaggtctcct
     1261 aaaagatgga aacccgattc cctcagcaat cgcagcaaac tccggcatct acagcgctgg
     1321 aggaggtgga agcggaggag gaggaagcgg aggaggaggt agccccaaga agaagagaaa
     1381 ggtggaggcc agcggtggag gaagtggagg tggttcaggt ggaggatccg gcgaagctgc
     1441 accctgtgac tgtgatggag gtacacaaaa agaaaaaggc ccatattata cacaccttgg
     1501 ggcaggacca agtgtggctg ctgtcaggga gctcatggag actaggtttg gccagaaggg
     1561 gaaggcaatc cggattgaga agatagtgtt cacggggaag gaagggaaga gctctcaggg
     1621 ctgcccggtc gccaagtggg tgatcagaag aagtggtcct gaagagaagc ttatttgttt
     1681 ggttcgtgag cgtgtagacc atcactgttc gacggctgtg atagttgtcc ttatcctgct
     1741 gtgggaaggt atccctcgcc tgatggctga ccgcctgtac aaagagctca ctgagaactt
     1801 gaggtcctac agcggacatc ccacagaccg aagatgtacc ctcaacaaaa agcgtacctg
     1861 cacctgtcaa ggcatcgacc caaaaacctg cggagcgtcc ttctcctttg gctgttcgtg
     1921 gagcatgtat ttcaacggct gtaagtttgg gaggagtgaa aaccccagaa aattcagact
     1981 tgctccaaac taccccttac ataactacta taagagaatt actggaatga gttctgaagg
     2041 aagtgacgtg aaaaccgggt ggatcattcc agaccgcaag accctcataa gcagagagga
     2101 aaaacagctt gaaaagaatt tacaagaatt ggctacagta ttagctccac tttacaagca
     2161 gatggctcca gttgcttatc aaaatcaggt ggaatatgaa gaagttgctg gagactgtcg
     2221 acttggaaat gaagaggggc gtcctttctc tggtgtcacc tgttgcatgg atttttgtgc
     2281 ccattctcac aaggacattc acaacatgca caacggaagc accgtggtgt gtacgttgat
     2341 tcgagcagat ggccgtgaca caaattgtcc cgaggatgaa caactccacg tcctgccact
     2401 ataccggctt gcagacactg atgaatttgg ctccgtggaa gggatgaagg ccaaaatcaa
     2461 atctggggcc atccaagtca atgggccaac caggaagagg cgactacgtt ttactgagcc
     2521 tgttcctcga tgtgggaaga gggccaaaat gaagcagaac cacaataaat caggttcaca
     2581 caacactaag agcttttcat cagcctcatc tacttctcac ctagtgaaag acgaatctac
     2641 agacttctgt cccctgcagg cttcctccgc agaaacatct acctgtacgt acagtaaaac
     2701 agcctcaggt gggtttgcag aaacaagtag tattctccac tgcacaatgc cttctggagc
     2761 acacagtggt gctaatgcag ctgctgggga atgtactgga acggtgcagc ctgccgaggt
     2821 ggctgctcat cctcaccagt ctcttcccac agccgattct cccgttcatg ctgagcctct
     2881 cactagtcca tctgagcagc taacttctaa ccagtcaaac cagcagctcc ctctcctcag
     2941 caattctcag aaactggctt cctgtcaggt ggaagatgag cggcaccctg aagcggatga
     3001 gcctcagcac cccgaggacg ataacttgcc tcaacttgat gagttctggt cagacagtga
     3061 ggagatctac gccgatcctt cctttggtgg cgtggcgata gcacccattc acggctcggt
     3121 gctcattgag tgcgctcgga aggagcttca tgctaccacc tctttgcgct cccccaaacg
     3181 aggggtccct tttcgtgtgt cccttgtatt ctaccagcac aaaagcctaa acaagcctaa
     3241 tcatggtttt gatatcaaca aaattaagtg taaatgcaaa aaagtaacga aaaaaaagcc
     3301 cgcagaccgg gagtgtcctg atgtatcccc cgaagccaat ttatcacacc aaattccttc
     3361 tcgagttgca tcaaccttaa cccgagacaa tgttgttacc gtgtccccat actctctcac
     3421 tcatgttgcg ggaccctaca atcgttgggt cgcggccgca gactacaagg acgatgacga
     3481 taagtaactc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     3541 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3601 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3661 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3721 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     3781 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3841 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3901 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcggggcat
     3961 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     4021 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     4081 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     4141 ggtctattct tttgatttat aagggatttt ggggatttcg gcctattggt taaaaaatga
     4201 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     4261 ggaaagtccc caggctcccc aggcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4321 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4381 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     4441 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4501 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     4561 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt
     4621 gatgaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
     4681 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
     4741 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
     4801 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat
     4861 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
     4921 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga
     4981 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
     5041 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
     5101 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
     5161 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
     5221 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
     5281 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
     5341 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
     5401 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
     5461 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
     5521 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg
     5581 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
     5641 ggaatagcac gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     5701 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     5761 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5821 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5881 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     5941 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     6001 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     6061 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     6121 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     6181 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     6241 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     6301 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     6361 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     6421 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     6481 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     6541 tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     6601 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     6661 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6721 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6781 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6841 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6901 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6961 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     7021 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     7081 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     7141 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     7201 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     7261 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     7321 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7381 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     7441 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     7501 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     7561 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7621 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7681 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7741 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7801 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7861 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7921 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7981 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     8041 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     8101 cgtc
//
