LOCUS       Exported                6377 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  C-terminal tagging with mAID-mCherry2 using CRISPR/Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    pMK293 (mAID-mCherry2-Hygro)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6377)
  AUTHORS   Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT
  TITLE     Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron 
            Tagging with Short Homology Donors.
  JOURNAL   Cell Rep. 2016 Apr 5;15(1):210-8. doi: 10.1016/j.celrep.2016.03.001.
            Epub 2016 Mar 24.
  PUBMED    27052166
REFERENCE   2  (bases 1 to 6377)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6377
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(91..110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     301..322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             681..884
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             891..1601
                     /codon_start=1
                     /product="derivative of the monomeric red fluorescent 
                     protein mCherry"
                     /label=mCherry2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQCRTMGWEASTERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1036..1054)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    1719..1840
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1756..1775)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1810..1829
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1871..1904)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        1965..2464
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2000..2023)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2397..2416
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2525..3544
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     protein_bind    complement(4107..4140)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(4189..4209)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4189..4207)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4228..4244)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4228..4244)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4241..4263)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4252..4268
                     /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)."
     promoter        complement(4276..4306)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4321..4342
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4459..4476)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4630..5218)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4710..4729)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5389..6249)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6012..6031
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6250..6354)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctccc
      661 cgggatccgg tgcaggcgcc aaggagaaga gtgcttgtcc taaagatcca gccaaacctc
      721 cggccaaggc acaagttgtg ggatggccac cggtgagatc ataccggaag aacgtgatgg
      781 tttcctgcca aaaatcaagc ggtggcccgg aggcggcggc gttcgtgaag gtatcaatgg
      841 acggagcacc gtacttgagg aaaatcgatt tgaggatgta taaagctagc atggtgagca
      901 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
      961 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     1021 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     1081 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     1141 tccccgacta cttgaagctg tccttccccg agggcttcaa ttgggagcgc gtgatgaact
     1201 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     1261 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagtgtc
     1321 gtaccatggg ctgggaggcc tccactgagc ggatgtaccc cgaggacggc gccctgaagg
     1381 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     1441 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc gacatcaagt
     1501 tggacatcct ttcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     1561 gccgccactc caccggcggc atggacgagc tgtacaagta atctagataa ctgatcataa
     1621 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     1681 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     1741 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     1801 attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtcgat catattcaat
     1861 aacccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtc tgaagaggag
     1921 tttacgtcca gccaagctta ggatctcgac ctcgaaattc taccgggtag gggaggcgct
     1981 tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac
     2041 aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac cgactccgtt
     2101 ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc
     2161 cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt
     2221 gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata
     2281 gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg
     2341 gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt
     2401 ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct
     2461 ttcgacctgc atccatctag atctcgagca gctgaagctt accgctagca tggatagatc
     2521 cggaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
     2581 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
     2641 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
     2701 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
     2761 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
     2821 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc
     2881 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
     2941 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
     3001 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
     3061 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
     3121 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
     3181 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
     3241 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
     3301 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
     3361 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
     3421 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
     3481 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
     3541 ataggctagc cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca
     3601 tgcatcgatg atatcagatc cccgggatgc agaaattgat gatctattaa acaataaaga
     3661 tgtccactaa aatggaagtt tttcctgtca tactttgtta agaagggtga gaacagagta
     3721 cctacatttt gaatggaagg attggagcta cgggggtggg ggtggggtgg gattagataa
     3781 atgcctgctc tttactgaag gctctttact attgctttat gataatgttt catagttgga
     3841 tatcataatt taaacaagca aaaccaaatt aagggccagc tcattcctcc cactcatgat
     3901 ctatagatct atagatctct cgtgggatca ttgtttttct cttgattccc actttgtggt
     3961 tctaagtact gtggtttcca aatgtgtcag tttcatagcc tgaagaacga gatcagcagc
     4021 ctctgttcca catacacttc attctcagta ttgttttgcc aagttctaat tccatcagaa
     4081 gctggtcgag atccggaacc cttaatataa cttcgtataa tgtatgctat acgaagttat
     4141 taggtccctc gaagaggttc actaggatcc ggtacccagc ttttgttccc tttagtgagg
     4201 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4261 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4321 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4381 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4441 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4501 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4561 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4621 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4681 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4741 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4801 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4861 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4921 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4981 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5041 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5101 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5161 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5221 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5281 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5341 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5401 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5461 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5521 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5581 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5641 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5701 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5761 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5821 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5881 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5941 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6001 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6061 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6121 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6181 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6241 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6301 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6361 tccccgaaaa gtgccac
//
