LOCUS       Exported                6117 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  C-terminal tagging with mAID-mClover using CRISPR/Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    pMK289 (mAID-mClover-NeoR)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6117)
  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 6117)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6117
                     /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..1610
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(936..957)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1544..1565
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1728..1849
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1765..1784)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1819..1838
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1856..2311)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2338..2442
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2444..2801
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(2467..2487)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2652..2787
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2714..2733
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2836..3630
                     /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(2890..2909)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3500..3519
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3818..3837)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     primer_bind     complement(3929..3949)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3929..3947)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3968..3984)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3968..3984)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3981..4003)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3992..4008
                     /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(4016..4046)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4061..4082
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4199..4216)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4370..4958)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4450..4469)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5129..5989)
                     /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     5752..5771
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5990..6094)
                     /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 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
      961 acggccacaa gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac ggcaagctga
     1021 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     1081 ccttcggcta cggcgtggcc tgcttcagcc gctaccccga ccacatgaag cagcacgact
     1141 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctct ttcaaggacg
     1201 acggtaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1261 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1321 acaacttcaa cagccacaac gtctatatca cggccgacaa gcagaagaac ggcatcaagg
     1381 ctaacttcaa gatccgccac aacgttgagg acggcagcgt gcagctcgcc gaccactacc
     1441 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagcc
     1501 atcagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1561 tcgtgaccgc cgccgggatt acacatggca tggacgagct gtacaagtaa tctagataac
     1621 tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     1681 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     1741 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     1801 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt
     1861 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     1921 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     1981 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     2041 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     2101 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     2161 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     2221 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     2281 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     2341 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2401 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     2461 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2521 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     2581 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     2641 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     2701 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     2761 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga
     2821 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     2881 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     2941 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3001 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3061 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3121 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3181 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3241 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3301 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3361 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3421 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3481 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3541 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     3601 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     3661 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     3721 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     3781 tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac
     3841 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt
     3901 cgttggatcc ggtacccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     3961 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4021 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     4081 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4141 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4201 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4261 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4321 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4381 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4441 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4501 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4561 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4621 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4681 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4741 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4801 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4861 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4921 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4981 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5041 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5101 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5161 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5221 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5281 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5341 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5401 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5461 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5521 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5581 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5641 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5701 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5761 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5821 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5881 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5941 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6001 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6061 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
