LOCUS       Exported                6108 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  C-terminal tagging with mAID-mCherry2 using CRISPR/Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    pMK292 (mAID-mCherry2-NeoR)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6108)
  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 6108)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6108
                     /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     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"
     rep_origin      complement(1847..2302)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2329..2433
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2435..2792
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(2458..2478)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2643..2778
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2705..2724
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2827..3621
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2881..2900)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3491..3510
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3809..3828)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     primer_bind     complement(3920..3940)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3920..3938)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3959..3975)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3959..3975)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3972..3994)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3983..3999
                     /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(4007..4037)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4052..4073
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4190..4207)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4361..4949)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4441..4460)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5120..5980)
                     /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     5743..5762
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5981..6085)
                     /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 acgcgtaaat tgtaagcgtt
     1861 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     1921 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     1981 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     2041 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     2101 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     2161 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     2221 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     2281 aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     2341 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2401 taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga
     2461 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2521 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     2581 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     2641 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     2701 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     2761 gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg
     2821 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     2881 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     2941 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     3001 gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     3061 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     3121 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     3181 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     3241 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     3301 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg
     3361 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     3421 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     3481 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     3541 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     3601 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     3661 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     3721 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     3781 tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     3841 cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgttggatc
     3901 cggtacccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt
     3961 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4021 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4081 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4141 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4201 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4261 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4321 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4381 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4441 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4501 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4561 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4621 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4681 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4741 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4801 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4861 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4921 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4981 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5041 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5101 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5161 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5221 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5281 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5341 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5401 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5461 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5521 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5581 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5641 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5701 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5761 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5821 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5881 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5941 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6001 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6061 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
