LOCUS CRISPR-SP-Cas9 reporter 8960 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Mammalian CRISPR-SP-Cas9 reporter.
ACCESSION .
VERSION .
KEYWORDS CRISPR-SP-Cas9 reporter
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8960)
AUTHORS D'Astolfo DS, Pagliero RJ, Pras A, Karthaus WR, Clevers H, Prasad V,
Lebbink RJ, Rehmann H, Geijsen N
TITLE Efficient Intracellular Delivery of Native Proteins.
JOURNAL Cell. 2015 Apr 23;161(3):674-690. doi: 10.1016/j.cell.2015.03.028.
PUBMED 25910214
REFERENCE 2 (bases 1 to 8960)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8960
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 1..634
/label=3' LTR
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1721..1765
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
misc_feature 2058..2175
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2223..3401
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 2454..3392
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 3349..3369
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
regulatory 3406..3415
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 3421..3447
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 3503..4201
/codon_start=1
/product="dimeric variant of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=dTomato
/note="mammalian codon-optimized"
/translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK
LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL
VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ
ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH
LFLYGMDELYK"
primer_bind complement(3633..3651)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 3901..3920
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind 4095..4118
/label=DsRed1-C
/note="DsRed1, forward primer"
misc_feature 4208..4775
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
misc_feature 4225..4777
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
primer_bind complement(4369..4386)
/label=IRES reverse
/note="IRES internal ribosome entry site, reverse primer.
Also called pCDH-rev"
primer_bind 4596..4615
/label=IRES-F
/note="IRES internal ribosome entry site, forward primer"
CDS 4789..5388
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(4789..4808)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 5285..5305
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
misc_feature 5402..5990
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(5455..5475)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(5873..5884)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 6138..6318
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(6365..6383)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 6451..6555
/gene="bla"
/label=AmpR promoter
CDS 6556..7416
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(6774..6793)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 7587..8175
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 8076..8095
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 8329..8346
/label=L4440
/note="L4440 vector, forward primer"
primer_bind 8438..8457
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
protein_bind 8586..8607
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 8622..8652
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 8660..8676
/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)."
primer_bind 8665..8687
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 8684..8700
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 8684..8700
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 8750..8769
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
ORIGIN
1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagtcgt
2221 gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
2281 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
2341 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
2401 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg
2461 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa
2521 ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt
2581 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct
2641 ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg
2701 ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct
2761 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg
2821 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc
2881 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc
2941 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac
3001 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga
3061 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc
3121 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg
3181 attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga
3241 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt
3301 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga
3361 cagtggttca aagttttttt cttccatttc aggtgtcgtg aagcggccgc catgggatct
3421 tacccttatg atgtgcctga ctacgccgga ggaggaggat ctggggccac tagggacagg
3481 atcggagctc cggccgcata aggtgagcaa gggcgaggag gtcatcaaag agttcatgcg
3541 cttcaaggtg cgcatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg
3601 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc
3661 cctgcccttc gcctgggaca tcctgtcccc ccagttcatg tacggctcca aggcgtacgt
3721 gaagcacccc gccgacatcc ccgattacaa gaagctgtcc ttccccgagg gcttcaagtg
3781 ggagcgcgtg atgaacttcg aggacggcgg tctggtgacc gtgacccagg actcctccct
3841 gcaggacggc acgctgatct acaaggtgaa gatgcgcggc accaacttcc cccccgacgg
3901 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc accgagcgcc tgtacccccg
3961 cgacggcgtg ctgaagggcg agatccacca ggccctgaag ctgaaggacg gcggccacta
4021 cctggtggag ttcaagacca tctacatggc caagaagccc gtgcaactgc ccggctacta
4081 ctacgtggac accaagctgg acatcacctc ccacaacgag gactacacca tcgtggaaca
4141 gtacgagcgc tccgagggcc gccaccacct gttcctgtac ggcatggacg agctgtacaa
4201 gtaaggatcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt
4261 gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc
4321 ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag
4381 gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac
4441 aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc
4501 tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc
4561 acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca
4621 aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt
4681 gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg
4741 gggacgtggt tttcctttga aaaacacgat gataatatgg ccacacatat gaccgagtac
4801 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
4861 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
4921 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
4981 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
5041 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
5101 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
5161 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
5221 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
5281 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
5341 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaat cgatagatcc
5401 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
5461 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
5521 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
5581 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
5641 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
5701 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
5761 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
5821 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
5881 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
5941 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc ctggtacctt
6001 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
6061 gactggaagg gctaattcac tcccaacgaa gacaagatca cctgcaggac aggcgcgccc
6121 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
6181 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
6241 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
6301 tgtggaaaat ctctagcacc cgggcgatta aggaaagggc tagatcattc ttgaagacga
6361 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
6421 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
6481 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
6541 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
6601 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
6661 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
6721 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
6781 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
6841 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
6901 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
6961 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
7021 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
7081 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
7141 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
7201 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
7261 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
7321 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
7381 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
7441 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
7501 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
7561 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
7621 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
7681 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
7741 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
7801 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
7861 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
7921 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
7981 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
8041 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
8101 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
8161 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
8221 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
8281 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
8341 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
8401 cattaatgca gcaagctcat ggctgactaa ttttttttat ttatgcagag gccgaggccg
8461 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
8521 gcaaaaagct ccccgtggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
8581 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
8641 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
8701 atgattacga atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
8761 aaactcatca atgtatctta tcatgtctgg atcaactgga taactcaagc taaccaaaat
8821 catcccaaac ttcccacccc ataccctatt accactgcca attacctgtg gtttcattta
8881 ctctaaacct gtgattcctc tgaattattt tcattttaaa gaaattgtat ttgttaaata
8941 tgtactacaa acttagtagt
//
