LOCUS PE2-VRQR 9753 bp DNA circular SYN 22-MAY-2020
DEFINITION Prime editing in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS pCMV-PE2.
SOURCE synthetic DNA construct.
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9753)
TITLE Liu Lab plasmids
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 9753)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Oct 18, 2019 from SnapGene Server 1.1.58
http://www.snapgene.com
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http://www.invitrogen.com/
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COMMENT LSOWNER|
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FEATURES Location/Qualifiers
source 1..9753
/organism="synthetic DNA construct"
/mol_type="other DNA"
/vntifkey="98"
enhancer 1..380
/vntifkey="9"
/label=CMV\enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 381..584
/vntifkey="30"
/label=CMV\promoter
/note="human cytomegalovirus (CMV) immediate early promoter"
primer_bind 535..555
/vntifkey="28"
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 626..645
/vntifkey="28"
/label=T7
/note="T7 promoter, forward primer"
promoter 626..644
/vntifkey="30"
/label=T7\promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 694..714
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/vntifkey="4"
/label=SV40\NLS
CDS 715..4815
/codon_start=1
/product="nickase mutant of the Cas9 endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system"
/vntifkey="4"
/label=Cas9(H840A)
/note="generates RNA-guided single strand nicks in DNA due to the H840A mutation in the HNH catalytic domain"
CDS 6988..7008
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/vntifkey="4"
/label=SV40\NLS
CDS 7017..7034
/codon_start=1
/product="6xHis affinity tag"
/vntifkey="4"
/label=6xHis
primer_bind complement(7057..7074)
/vntifkey="28"
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse"
polyA_signal 7063..7287
/vntifkey="25"
/label=bGH\poly(A)\signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(7358..7374)
/vntifkey="28"
/label=M13\rev
/note="common sequencing primer, one of multiple similar variants"
primer_bind complement(7358..7374)
/vntifkey="28"
/label=M13\Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(7371..7393)
/vntifkey="28"
/label=M13/pUC\Reverse
/note="In lacZ gene"
protein_bind 7382..7398
/bound_moiety="lac repressor encoded by lacI"
/vntifkey="31"
/label=lac\operator
/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(7406..7436)
/vntifkey="30"
/label=lac\promoter
/note="promoter for the E. coli lac operon"
protein_bind 7451..7472
/bound_moiety="E. coli catabolite activator protein"
/vntifkey="31"
/label=CAP\binding\site
/note="CAP binding activates transcription in the presence of cAMP."
primer_bind complement(7589..7606)
/vntifkey="28"
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(7760..8348)
/direction=LEFT
/vntifkey="33"
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
primer_bind complement(7840..7859)
/vntifkey="28"
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(8519..9379)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/vntifkey="4"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and related antibiotics"
primer_bind 9142..9161
/vntifkey="28"
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(9380..9484)
/gene="bla"
/vntifkey="30"
/label=AmpR\promoter
primer_bind complement(9563..9582)
/vntifkey="28"
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable marker"
overhang 3229..3231
/vntifkey="274"
/label=H840A
overhang 2865..2883
/vntifkey="274"
/label=PE_1F
overhang complement(5235..5255)
/vntifkey="274"
/label=PE_2R
misc_feature 3376..4794
/vntifkey="21"
/label=from\VRQR
BASE COUNT 2616 a 2654 c 2575 g 1908 t
ORIGIN
1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
601 agatccgcta gagatccgcg gccgctaata cgactcacta tagggagagc cgccaccatg
661 aaacggacag ccgacggaag cgagttcgag tcaccaaaga agaagcggaa agtcgacaag
721 aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac
781 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc
841 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg
901 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa
961 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag
1021 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg
1081 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg
1141 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag
1201 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag
1261 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc
1321 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa
1381 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc
1441 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa
1501 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc
1561 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc
1621 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag
1681 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg
1741 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt
1801 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg
1861 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg
1921 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg
1981 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc
2041 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc
2101 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac
2161 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc
2221 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag
2281 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag
2341 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag
2401 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg
2461 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac
2521 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg
2581 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc
2641 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc
2701 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg
2761 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc
2821 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac
2881 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg
2941 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg
3001 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg
3061 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac
3121 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac
3181 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacgc tatcgtgcct
3241 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac
3301 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg
3361 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc
3421 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa
3481 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac
3541 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg
3601 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac
3661 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag
3721 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc
3781 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg
3841 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc
3901 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg
3961 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc
4021 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag
4081 aaggactggg accctaagaa gtacggcggc ttcgtgagcc ccaccgtggc ctattctgtg
4141 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg
4201 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa
4261 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg
4321 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccagagaact gcagaaggga
4381 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag
4441 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag
4501 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc
4561 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga
4621 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc
4681 ttcaagtact ttgacaccac catcgaccgg aagcagtaca gaagcaccaa agaggtgctg
4741 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct
4801 cagctgggag gtgactctgg aggatctagc ggaggatcct ctggcagcga gacaccagga
4861 acaagcgagt cagcaacacc agagagcagt ggcggcagca gcggcggcag cagcacccta
4921 aatatagaag atgagtatcg gctacatgag acctcaaaag agccagatgt ttctctaggg
4981 tccacatggc tgtctgattt tcctcaggcc tgggcggaaa ccgggggcat gggactggca
5041 gttcgccaag ctcctctgat catacctctg aaagcaacct ctacccccgt gtccataaaa
5101 caatacccca tgtcacaaga agccagactg gggatcaagc cccacataca gagactgttg
5161 gaccagggaa tactggtacc ctgccagtcc ccctggaaca cgcccctgct acccgttaag
5221 aaaccaggga ctaatgatta taggcctgtc caggatctga gagaagtcaa caagcgggtg
5281 gaagacatcc accccaccgt gcccaaccct tacaacctct tgagcgggct cccaccgtcc
5341 caccagtggt acactgtgct tgatttaaag gatgcctttt tctgcctgag actccacccc
5401 accagtcagc ctctcttcgc ctttgagtgg agagatccag agatgggaat ctcaggacaa
5461 ttgacctgga ccagactccc acagggtttc aaaaacagtc ccaccctgtt taatgaggca
5521 ctgcacagag acctagcaga cttccggatc cagcacccag acttgatcct gctacagtac
5581 gtggatgact tactgctggc cgccacttct gagctagact gccaacaagg tactcgggcc
5641 ctgttacaaa ccctagggaa cctcgggtat cgggcctcgg ccaagaaagc ccaaatttgc
5701 cagaaacagg tcaagtatct ggggtatctt ctaaaagagg gtcagagatg gctgactgag
5761 gccagaaaag agactgtgat ggggcagcct actccgaaga cccctcgaca actaagggag
5821 ttcctaggga aggcaggctt ctgtcgcctc ttcatccctg ggtttgcaga aatggcagcc
5881 cccctgtacc ctctcaccaa accggggact ctgtttaatt ggggcccaga ccaacaaaag
5941 gcctatcaag aaatcaagca agctcttcta actgccccag ccctggggtt gccagatttg
6001 actaagccct ttgaactctt tgtcgacgag aagcagggct acgccaaagg tgtcctaacg
6061 caaaaactgg gaccttggcg tcggccggtg gcctacctgt ccaaaaagct agacccagta
6121 gcagctgggt ggcccccttg cctacggatg gtagcagcca ttgccgtact gacaaaggat
6181 gcaggcaagc taaccatggg acagccacta gtcattctgg ccccccatgc agtagaggca
6241 ctagtcaaac aaccccccga ccgctggctt tccaacgccc ggatgactca ctatcaggcc
6301 ttgcttttgg acacggaccg ggtccagttc ggaccggtgg tagccctgaa cccggctacg
6361 ctgctcccac tgcctgagga agggctgcaa cacaactgcc ttgatatcct ggccgaagcc
6421 cacggaaccc gacccgacct aacggaccag ccgctcccag acgccgacca cacctggtac
6481 acggatggaa gcagtctctt acaagaggga cagcgtaagg cgggagctgc ggtgaccacc
6541 gagaccgagg taatctgggc taaagccctg ccagccggga catccgctca gcgggctgaa
6601 ctgatagcac tcacccaggc cctaaagatg gcagaaggta agaagctaaa tgtttatact
6661 gatagccgtt atgcttttgc tactgcccat atccatggag aaatatacag aaggcgtggg
6721 tggctcacat cagaaggcaa agagatcaaa aataaagacg agatcttggc cctactaaaa
6781 gccctctttc tgcccaaaag acttagcata atccattgtc caggacatca aaagggacac
6841 agcgccgagg ctagaggcaa ccggatggct gaccaagcgg cccgaaaggc agccatcaca
6901 gagactccag acacctctac cctcctcata gaaaattcat caccctctgg cggctcaaaa
6961 agaaccgccg acggcagcga attcgagccc aagaagaaga ggaaagtcta accggtcatc
7021 atcaccatca ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
7081 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
7141 ctgtcctttc ctaataaaat gagaaaattg catcgcattg tctgagtagg tgtcattcta
7201 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
7261 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga
7321 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
7381 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
7441 agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
7501 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
7561 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
7621 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
7681 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
7741 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
7801 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
7861 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
7921 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
7981 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
8041 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
8101 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
8161 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
8221 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
8281 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
8341 gatctcaaga agatcctttg atcttttcta cggggtctga cactcagtgg aacgaaaact
8401 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
8461 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
8521 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
8581 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
8641 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
8701 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
8761 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
8821 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
8881 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
8941 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
9001 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
9061 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
9121 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
9181 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
9241 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
9301 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
9361 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
9421 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
9481 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg
9541 atcccctagg gtctactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
9601 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
9661 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
9721 tgcttcgcga tgtacgggcc agatatacgc gtt
//