LOCUS pMK283 (Stag-3FLAG-Neo) 5335 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION C-terminal tagging with Stag-3FLAG using CRIPSR/Cas9.
ACCESSION .
VERSION .
KEYWORDS pMK283 (Stag-3FLAG-Neo)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5335)
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 5335)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..5335
/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 677..721
/codon_start=1
/product="affinity and epitope tag derived from pancreatic
ribonuclease A"
/label=S-Tag
/translation="KETAAAKFERQHMDS"
CDS 734..799
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
polyA_signal 931..1052
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(968..987)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 1022..1041
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin complement(1059..1514)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 1541..1645
/gene="bla"
/label=AmpR promoter
promoter 1647..2004
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(1670..1690)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 1855..1990
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 1917..1936
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 2039..2833
/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(2093..2112)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 2703..2722
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind complement(3021..3040)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
primer_bind complement(3147..3167)
/label=T3
/note="T3 promoter, forward primer"
promoter complement(3147..3165)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(3186..3202)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3186..3202)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(3199..3221)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 3210..3226
/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(3234..3264)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3279..3300
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(3417..3434)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(3588..4176)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(3668..3687)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(4347..5207)
/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 4970..4989
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(5208..5312)
/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 tggagctcgg
661 atccggtgca ggcgccaagg aaaccgccgc tgcaaaattc gaacgccagc acatggacag
721 ccgatcccgg gctgactaca aagaccatga cggtgattat aaagatcatg acatcgacta
781 caaggatgac gatgacaagt agtgtacaag taatctagat aactgatcat aatcagccat
841 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
901 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
961 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
1021 tgtggtttgt ccaaactcat caatgtatct taacgcgtaa attgtaagcg ttaatatttt
1081 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
1141 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
1201 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
1261 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
1321 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
1381 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
1441 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
1501 gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
1561 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
1621 tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt
1681 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
1741 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
1801 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
1861 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
1921 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
1981 tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat
2041 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
2101 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
2161 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
2221 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
2281 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
2341 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
2401 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
2461 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
2521 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg
2581 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
2641 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
2701 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
2761 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
2821 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
2881 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
2941 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
3001 caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
3061 atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgttgga tccctcgagg
3121 gggggcccgg tacccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
3181 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
3241 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
3301 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
3361 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
3421 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
3481 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
3541 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
3601 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
3661 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
3721 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
3781 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
3841 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
3901 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
3961 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
4021 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
4081 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
4141 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
4201 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
4261 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
4321 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
4381 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
4441 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
4501 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
4561 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
4621 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
4681 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
4741 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
4801 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
4861 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
4921 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
4981 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
5041 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
5101 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
5161 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
5221 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
5281 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccac
//
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