LOCUS pMK284 (Stag-3FLAG-Hygro) 5589 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION C-terminal tagging with Stag-3FLAG using CRIPSR/Cas9.
ACCESSION .
VERSION .
KEYWORDS pMK284 (Stag-3FLAG-Hygro)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5589)
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 5589)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..5589
/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 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"
protein_bind complement(1083..1116)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
promoter 1177..1676
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
primer_bind complement(1212..1235)
/label=MSCV-rev
/note="Murine stem cell virus, reverse primer"
primer_bind 1609..1628
/label=mPGK-F
/note="Mouse PGK promoter, forward primer"
CDS 1737..2756
/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(3319..3352)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
primer_bind complement(3401..3421)
/label=T3
/note="T3 promoter, forward primer"
promoter complement(3401..3419)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(3440..3456)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3440..3456)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(3453..3475)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 3464..3480
/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(3488..3518)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3533..3554
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(3671..3688)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(3842..4430)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(3922..3941)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(4601..5461)
/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 5224..5243
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(5462..5566)
/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 taacgcgtcg atcatattca ataaccctta
1081 atataacttc gtataatgta tgctatacga agttattagg tctgaagagg agtttacgtc
1141 cagccaagct taggatctcg acctcgaaat tctaccgggt aggggaggcg cttttcccaa
1201 ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc
1261 tctggcctcg cacacattcc acatccaccg gtaggcgcca accgactccg ttctttggtg
1321 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc
1381 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg
1441 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt
1501 gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg
1561 gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc
1621 ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgacct
1681 gcatccatct agatctcgag cagctgaagc ttaccgctag catggataga tccggaaagc
1741 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
1801 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
1861 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
1921 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
1981 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
2041 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
2101 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
2161 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
2221 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
2281 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
2341 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
2401 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
2461 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
2521 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
2581 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
2641 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
2701 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaataggcta
2761 gccgcccgcc ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc catgcatcga
2821 tgatatcaga tccccgggat gcagaaattg atgatctatt aaacaataaa gatgtccact
2881 aaaatggaag tttttcctgt catactttgt taagaagggt gagaacagag tacctacatt
2941 ttgaatggaa ggattggagc tacgggggtg ggggtggggt gggattagat aaatgcctgc
3001 tctttactga aggctcttta ctattgcttt atgataatgt ttcatagttg gatatcataa
3061 tttaaacaag caaaaccaaa ttaagggcca gctcattcct cccactcatg atctatagat
3121 ctatagatct ctcgtgggat cattgttttt ctcttgattc ccactttgtg gttctaagta
3181 ctgtggtttc caaatgtgtc agtttcatag cctgaagaac gagatcagca gcctctgttc
3241 cacatacact tcattctcag tattgttttg ccaagttcta attccatcag aagctggtcg
3301 agatccggaa cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtccc
3361 tcgaagaggt tcactaggat ccggtaccca gcttttgttc cctttagtga gggttaattg
3421 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
3481 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
3541 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
3601 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
3661 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
3721 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
3781 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
3841 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
3901 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
3961 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
4021 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
4081 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
4141 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
4201 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
4261 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
4321 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
4381 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
4441 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
4501 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
4561 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
4621 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
4681 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
4741 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
4801 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
4861 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
4921 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
4981 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
5041 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
5101 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
5161 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
5221 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
5281 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
5341 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
5401 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
5461 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
5521 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
5581 aagtgccac
//
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