LOCUS Cas12f-GE ver4.0 6832 bp ds-DNA circular SYN 02-NOV-2021
DEFINITION Expresses Cas12f-GE in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS Cas12f-GE ver4.0
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6832)
AUTHORS Kim DY, Lee JM, Moon SB, Chin HJ, Park S, Lim Y, Kim D, Koo T, Ko
JH, Kim YS
TITLE Efficient CRISPR editing with a hypercompact Cas12f1 and engineered
guide RNAs delivered by adeno-associated virus.
JOURNAL Nat Biotechnol. 2021 Sep 2. pii: 10.1038/s41587-021-01009-z. doi:
10.1038/s41587-021-01009-z.
PUBMED 34475560
REFERENCE 2 (bases 1 to 6832)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Nov 2, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6832
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 6..246
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 6..26
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 177..196
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
enhancer 572..857
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 859..1136
/label=chicken beta-actin promoter
intron 1137..1364
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
regulatory 1376..1385
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1388..1408
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3020..3067
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 3083..3136
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 3137..3850
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
CDS 3137..3850
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
primer_bind complement(3179..3200)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(3440..3459)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 3787..3808
/label=EGFP-C
/note="EGFP, forward primer"
primer_bind complement(3878..3895)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 3884..4091
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 4100..4240
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4100..4229
/label=AAV2 ITR
rep_origin 4315..4770
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4402..4421)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4612..4633
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4787..4806)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4906..4928
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4966..4984)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5052..5156
/gene="bla"
/label=AmpR promoter
CDS 5157..6017
/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(5375..5394)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6188..6776
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6677..6696
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 catgtgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
61 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
121 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
181 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
241 aaggacgaaa caccgaccgc ttcaccaaaa gctgtccctt aggggattag aacttgagtg
301 aaggtgggct gcttgcatca gcctaatgtc gagaagtgct ttcttcggaa agtaaccctc
361 gaaacaaaga aaggaatgca acgagtcttc gacgcacctg acgtctaaga aaccattatt
421 atcatgacat taacctataa aaataggcgt atcacggatc ccgaagacga ttttattttt
481 tgttttagag ctagaaatag taagttaaaa taaggctagt ccgtttttag cgcgtgcgcc
541 aattctgcag acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc
601 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt
661 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
721 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
781 attgtgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
841 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
901 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
961 gggcgggggg gggggggggg cgcgcgccrg gsggggsggg gsggggsgrg gggsggggsg
1021 gggsgrggcg gagaggkgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
1081 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
1141 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
1201 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
1261 gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa
1321 tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac
1381 catggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccatggccaa
1441 gaacacaatt acaaagacac tgaagctgag gatcgtgaga ccatacaaca gcgctgaggt
1501 cgagaagatt gtggctgatg aaaagaacaa cagggaaaag atcgccctcg agaagaacaa
1561 ggataaggtg aaggaggcct gctctaagca cctgaaagtg gccgcctact gcaccacaca
1621 ggtggagagg aacgcctgtc tgttttgtaa agctcggaag ctggatgata agttttacca
1681 gaagctgcgg ggccagttcc ccgatgccgt cttttggcag gagattagcg agatcttcag
1741 acagctgcag aagcaggccg ccgagatcta caaccagagc ctgatcgagc tctactacga
1801 gatcttcatc aagggcaagg gcattgccaa cgcctcctcc gtggagcact acctgagcga
1861 cgtgtgctac acaagagccg ccgagctctt taagaacgcc gctatcgctt ccgggctgag
1921 gagcaagatt aagagtaact tccggctcaa ggagctgaag aacatgaaga gcggcctgcc
1981 cactacaaag agcgacaact tcccaattcc actggtgaag cagaaggggg gccagtacac
2041 agggttcgag atttccaacc acaacagcga ctttattatt aagatcccct ttggcaggtg
2101 gcaggtcaag aaggagattg acaagtacag gccctgggag aagtttgatt tcgagcaggt
2161 gcagaagagc cccaagccta tttccctgct gctgtccaca cagcggcgga agaggaacaa
2221 ggggtggtct aaggatgagg ggaccgaggc cgagattaag aaagtgatga acggcgacta
2281 ccagacaagc tacatcgagg tcaagcgggg cagtaagatt ggcgagaaga gcgcctggat
2341 gctgaacctg agcattgacg tgccaaagat tgataagggc gtggatccca gcatcatcgg
2401 agggatcgat gtgggggtca agagccccct cgtgtgcgcc atcaacaacg ccttcagcag
2461 gtacagcatc tccgataacg acctgttcca ctttaacaag aagatgttcg cccggcggag
2521 gattttgctc aagaagaacc ggcacaagcg ggccggacac ggggccaaga acaagctcaa
2581 gcccatcact atcctgaccg agaagagcga gaggttcagg aagaagctca tcgagagatg
2641 ggcctgcgag atcgccgatt tctttattaa gaacaaggtc ggaacagtgc agatggagaa
2701 cctcgagagc atgaagagga aggaggattc ctacttcaac attcggctga gggggttctg
2761 gccctacgct gagatgcaga acaagattga gtttaagctg aagcagtacg ggattgagat
2821 ccggaaggtg gcccccaaca acaccagcaa gacctgcagc aagtgcgggc acctcaacaa
2881 ctacttcaac ttcgagtacc ggaagaagaa caagttccca cacttcaagt gcgagaagtg
2941 caactttaag gagaacgccg attacaacgc cgccctgaac atcagcaacc ctaagctgaa
3001 gagcactaag gaggagccca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa
3061 gaaaaaggaa ttcggcagtg gagagggcag aggaagtctg ctaacatgcg gtgacgtcga
3121 ggagaatcct ggcccagtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
3181 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
3241 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
3301 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
3361 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
3421 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
3481 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
3541 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
3601 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
3661 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
3721 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
3781 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
3841 gctgtacaag gaattctaac tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
3901 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
3961 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
4021 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg
4081 catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
4141 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
4201 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
4261 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
4321 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
4381 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
4441 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
4501 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
4561 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
4621 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
4681 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
4741 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
4801 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
4861 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
4921 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
4981 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
5041 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
5101 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
5161 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
5221 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
5281 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
5341 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
5401 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
5461 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
5521 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
5581 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
5641 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
5701 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
5761 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
5821 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg
5881 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
5941 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
6001 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
6061 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
6121 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aaaatcaaag
6181 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
6241 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
6301 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
6361 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
6421 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
6481 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
6541 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
6601 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
6661 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
6721 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
6781 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct ca
//
