LOCUS pCMV-AsCas12f1 6845 bp ds-DNA circular SYN 10-SEP-2021
DEFINITION AsCas12f1-based Human cell genome editing.
ACCESSION .
VERSION .
KEYWORDS pCMV-AsCas12f1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6845)
AUTHORS Wu Z, Zhang Y, Yu H, Pan D, Wang Y, Wang Y, Li F, Liu C, Nan H, Chen
W, Ji Q
TITLE Programmed genome editing by a miniature CRISPR-Cas12f nuclease.
JOURNAL Nat Chem Biol. 2021 Sep 2. pii: 10.1038/s41589-021-00868-6. doi:
10.1038/s41589-021-00868-6.
PUBMED 34475565
REFERENCE 2 (bases 1 to 6845)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 10, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6845
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 14..393
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 394..597
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 548..568
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 639..658
/label=T7
/note="T7 promoter, forward primer"
promoter 639..657
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 665..674
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 677..697
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1976..1996
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2005..2022
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(2045..2062)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 2051..2275
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(2346..2362)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2346..2362)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
promoter 2389..2493
/gene="bla"
/label=AmpR promoter
CDS 2494..3354
/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 complement(2712..2731)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 3525..4113
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4014..4033
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4267..4284
/label=L4440
/note="L4440 vector, forward primer"
polyA_signal 4532..4653
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4569..4588)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 4623..4642
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
CDS complement(4800..5399)
/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(4883..4903)
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
primer_bind 5380..5399
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
promoter complement(5421..5931)
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
primer_bind complement(5484..5502)
/label=hPGK-F
/note="Human PGK promoter, forward primer"
misc_feature 5980..6097
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter complement(6352..6592)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(6402..6421)
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
primer_bind complement(6572..6592)
/label=hU6-F
/note="Human U6 promoter, forward primer"
misc_feature 6671..6762
/label=pause site
/note="RNA polymerase II transcriptional pause signal from
the human alpha-2 globin gene"
primer_bind complement(6703..6722)
/label=RVprimer3
/note="pGL3 vector, forward primer"
ORIGIN
1 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt
601 ttagtgaacc gtcagatccg ctagagatcc gcggccgcta atacgactca ctatagggag
661 agccgccacc atggccccca agaagaagag gaaagtcatg atcaaggtgt acagatacga
721 gatcgtgaag cctctggacc tggactggaa ggagttcggc accatcctga gacagctgca
781 gcaggaaacc agattcgccc tgaataaggc cacacagctg gcctgggagt ggatgggctt
841 cagcagcgac tacaaggata accacggcga gtaccccaag agcaaggaca tcctgggcta
901 caccaacgtg cacggctacg cctaccacac catcaagaca aaggcctaca gactgaactc
961 tggaaatctg agccagacca tcaagagagc cacagacagg ttcaaggcct accagaagga
1021 gatcctgcgc ggcgacatgt ctatccccag ctacaagagg gacatccccc tggacctgat
1081 caaggagaac atctccgtga acaggatgaa tcacggcgac tacatcgcca gcctgtctct
1141 gctgagcaac cccgccaagc aggagatgaa cgtgaagaga aagatctccg tgatcatcat
1201 cgtgaggggc gccggcaaga ccatcatgga cagaatcctg tccggcgagt accaggtgag
1261 cgccagccag attatccacg acgaccggaa gaacaagtgg tacctgaaca tcagctacga
1321 cttcgagcca cagaccagag tgctggacct gaacaagatc atgggcattg acctgggcgt
1381 ggccgtggcc gtgtacatgg ccttccagca cacccccgcc aggtacaagc tggagggcgg
1441 cgagattgag aacttcagga ggcaggtgga gagccggcgc atctccatgc tgagacaggg
1501 caagtacgcc ggcggcgcca ggggcggcca cggcagagac aagagaatca agcccattga
1561 gcagctgagg gataagatcg ccaatttcag agacaccacc aatcaccggt acagcagata
1621 catcgtggac atggccatca aggagggctg cggcacaatc cagatggagg atctgacaaa
1681 catcagagac atcggcagca gattcctgca gaactggacc tactacgacc tgcagcagaa
1741 gatcatctac aaggccgagg aggccggcat caaagtgatc aagatcgacc cccagtacac
1801 cagccagaga tgctccgagt gcggcaacat cgactccggc aacagaatcg gccaggccat
1861 ctttaagtgc cgggcctgcg gctacgaggc caacgccgac tacaacgccg cccggaatat
1921 cgccatcccc aacatcgaca agatcatcgc cgagagcatt aagtctggtg gttctcccaa
1981 gaagaagagg aaagtctaac cggtcatcat caccatcacc attgagttta aacccgctga
2041 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
2101 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
2161 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
2221 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
2281 gaggcggaaa gaaccagctg gggctcgata ccgtcgacct ctagctagag cttggcgtaa
2341 tcatggtcat agctgtttcc tgcaggtggc acttttcggg gaaatgtgcg cggaacccct
2401 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
2461 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
2521 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
2581 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
2641 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
2701 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
2761 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
2821 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
2881 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
2941 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
3001 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
3061 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
3121 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
3181 gctgataaat ctggagccgg tgagcgtggg tcccgcggta tcattgcagc actggggcca
3241 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
3301 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
3361 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
3421 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
3481 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
3541 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
3601 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
3661 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
3721 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
3781 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
3841 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
3901 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
3961 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
4021 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
4081 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
4141 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
4201 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
4261 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgct gccggcacct gtcctacgag
4321 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
4381 gaaggagctg actgggttga aggctctcaa gggcatcggt cgacggatcc ttatcgattt
4441 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
4501 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
4561 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
4621 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgctcgaagc ggccggccgc
4681 cccgactcta gaaagtggct aagatctaca gctgccttgt aagtcattgg tcttaaaggt
4741 accgatgcat ggggtcgtgc gctcctttcg gtcgggcgct gcgggtcgtg gggcgggcgt
4801 caggcaccgg gcttgcgggt catgcaccag gtgcgcggtc cttcgggcac ctcgacgtcg
4861 gcggtgacgg tgaagccgag ccgctcgtag aaggggaggt tgcggggcgc ggaggtttcc
4921 aggaaggcgg gcaccccggc gcgctcggcc gcctccactc cggggagcac gacggcgctg
4981 cccagaccct tgccctggtg gtcgggcgag acgccgacgg tggccaggaa ccacgcgggc
5041 tccttgggcc ggtgcggcgc caggaggcct tccatctgtt gctgcgcggc cagccgggaa
5101 ccgctcaact cggccatgcg cgggccgatc tcggcgaaca ccgcccccgc ttcgacgctc
5161 tccggcgtgg tccagaccgc caccgcggcg ccgtcgtccg cgacccacac cttgccgatg
5221 tcgagcccga cgcgcgtgag gaagagttct tgcagctcgg tgacccgctc gatgtggcgg
5281 tccggatcga cggtgtggcg cgtggcgggg tagtcggcga acgcggcggc gagggtgcgt
5341 acggccctgg ggacgtcgtc gcgggtggcg aggcgcaccg tgggcttgta ctcggtcatg
5401 gtaagctccg gtggatcccc ctggggagag aggtcggtga ttcggtcaac gagggagccg
5461 actgccgacg tgcgctccgg aggcttgcag aatgcggaac accgcgcggg caggaacagg
5521 gcccacacta ccgccccaca ccccgcctcc cgcaccgccc cttcccggcc gctgctctcg
5581 gcgcgccctg ctgagcagcc gctattggcc acagcccatc gcggtcggcg cgctgccatt
5641 gctccctggc gctgtccgtc tgcgagggta ctagtgagac gtgcggcttc cgtttgtcac
5701 gtccggcacg ccgcgaaccg caaggaacct tcccgactta ggggcggagc aggaagcgtc
5761 gccggggggc ccacaagggt agcggcgaag atccgggtga cgctgcgaac ggacgtgaag
5821 aatgtgcgag tcccagggtc ggcgccgctg cgtttcccgg aaccacgccc agagcagccg
5881 cgtccctgcg caaacccagg gctgccttgg aaaaggcgca accccaaccc cctcgagccg
5941 cggccaaagt ggatctctgc tgtccctgta ataaacccga aaattttgaa tttttgtaat
6001 ttgtttttgt aattctttag tttgtatgtc tgttgctatt atgtctacta ttctttcccc
6061 tgcactgtac cccccaatcc ccccttttct tttaaaattg tggatgaata ctgccatttg
6121 tctcgaggtc gagaattcaa aaaaaggaga ccgaccctct ctcgggtctc ggttcacact
6181 ccacaagcta gctcgcaaac ccttgtttat gagctatatg tgaagcgtcc gtgcggtctt
6241 tgtgcacgga gtttcagcct tttggctacc ttaccgagcg ttaccaaacc acttaacagt
6301 tttattggca cttctcggct tatcgtcgct gaaccgacga atcggtgttt cgtcctttcc
6361 acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc atatgatagt
6421 ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac tttctacgtc
6481 acgtattttg tactaatatc tttgtgttta cagtcaaatt aattccaatt atctctctaa
6541 cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc tcggtgaagg
6601 gggcggccgc tcgaggctag tctcgtgatc gataccgtcg agatccgttc actaatcggt
6661 acctatcgat agagaaatgt tctggcacct gcacttgcac tggggacagc ctattttgct
6721 agtttgtttt gtttcgtttt gttttgatgg agagcgtatg ttagtactat cgattcttga
6781 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
6841 gggcc
//
