LOCUS Cas12f-GE ver4.1 6802 bp ds-DNA circular SYN 18-OCT-2021
DEFINITION Expresses Cas12f-GE in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS Cas12f-GE ver4.1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6802)
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 6802)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Oct 18, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6802
/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 545..830
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
intron 1107..1334
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
regulatory 1346..1355
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1358..1378
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2990..3037
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 3053..3106
/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 3107..3820
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
CDS 3107..3820
/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(3149..3170)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(3410..3429)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 3757..3778
/label=EGFP-C
/note="EGFP, forward primer"
primer_bind complement(3848..3865)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 3854..4061
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 4070..4210
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4070..4199
/label=AAV2 ITR
rep_origin 4285..4740
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4372..4391)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4582..4603
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4757..4776)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4876..4898
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4936..4954)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5022..5126
/gene="bla"
/label=AmpR promoter
CDS 5127..5987
/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(5345..5364)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6158..6746
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6647..6666
/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 ttcacttaga gtgaaggtgg gctgcttgca tcagcctaat
301 gtcgagaagt gctttcttcg gaaagtaacc ctcgaaacaa agaaaggaat gcaacgagtc
361 ttcgacgcac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
421 cgtatcacgg atcccgaaga cgattttatt ttttgtttta gagctagaaa tagtaagtta
481 aaataaggct agtccgtttt tagcgcgtgc gccaattctg cagacaaatg gctctagagg
541 tacccgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
601 cattgacgtc aatagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
661 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
721 ttgacgtcaa tgacggtaaa tggcccgcct ggcattgtgc ccagtacatg accttatggg
781 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga
841 gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt
901 tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg cgcgcgccag
961 gcggggcggg gsggggsgrg gggsggggsg gggsgrggcg grgaggkgcg gcggcagcca
1021 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
1081 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc
1141 gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg
1201 tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt
1261 ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa
1321 tcactttttt tcaggttgga ccggtgccac catggcccca aagaagaagc ggaaggtcgg
1381 tatccacgga gtcccagcag ccatggccaa gaacacaatt acaaagacac tgaagctgag
1441 gatcgtgaga ccatacaaca gcgctgaggt cgagaagatt gtggctgatg aaaagaacaa
1501 cagggaaaag atcgccctcg agaagaacaa ggataaggtg aaggaggcct gctctaagca
1561 cctgaaagtg gccgcctact gcaccacaca ggtggagagg aacgcctgtc tgttttgtaa
1621 agctcggaag ctggatgata agttttacca gaagctgcgg ggccagttcc ccgatgccgt
1681 cttttggcag gagattagcg agatcttcag acagctgcag aagcaggccg ccgagatcta
1741 caaccagagc ctgatcgagc tctactacga gatcttcatc aagggcaagg gcattgccaa
1801 cgcctcctcc gtggagcact acctgagcga cgtgtgctac acaagagccg ccgagctctt
1861 taagaacgcc gctatcgctt ccgggctgag gagcaagatt aagagtaact tccggctcaa
1921 ggagctgaag aacatgaaga gcggcctgcc cactacaaag agcgacaact tcccaattcc
1981 actggtgaag cagaaggggg gccagtacac agggttcgag atttccaacc acaacagcga
2041 ctttattatt aagatcccct ttggcaggtg gcaggtcaag aaggagattg acaagtacag
2101 gccctgggag aagtttgatt tcgagcaggt gcagaagagc cccaagccta tttccctgct
2161 gctgtccaca cagcggcgga agaggaacaa ggggtggtct aaggatgagg ggaccgaggc
2221 cgagattaag aaagtgatga acggcgacta ccagacaagc tacatcgagg tcaagcgggg
2281 cagtaagatt ggcgagaaga gcgcctggat gctgaacctg agcattgacg tgccaaagat
2341 tgataagggc gtggatccca gcatcatcgg agggatcgat gtgggggtca agagccccct
2401 cgtgtgcgcc atcaacaacg ccttcagcag gtacagcatc tccgataacg acctgttcca
2461 ctttaacaag aagatgttcg cccggcggag gattttgctc aagaagaacc ggcacaagcg
2521 ggccggacac ggggccaaga acaagctcaa gcccatcact atcctgaccg agaagagcga
2581 gaggttcagg aagaagctca tcgagagatg ggcctgcgag atcgccgatt tctttattaa
2641 gaacaaggtc ggaacagtgc agatggagaa cctcgagagc atgaagagga aggaggattc
2701 ctacttcaac attcggctga gggggttctg gccctacgct gagatgcaga acaagattga
2761 gtttaagctg aagcagtacg ggattgagat ccggaaggtg gcccccaaca acaccagcaa
2821 gacctgcagc aagtgcgggc acctcaacaa ctacttcaac ttcgagtacc ggaagaagaa
2881 caagttccca cacttcaagt gcgagaagtg caactttaag gagaacgccg attacaacgc
2941 cgccctgaac atcagcaacc ctaagctgaa gagcactaag gaggagccca aaaggccggc
3001 ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggaa ttcggcagtg gagagggcag
3061 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccagtga gcaagggcga
3121 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
3181 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
3241 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
3301 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
3361 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
3421 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
3481 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
3541 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
3601 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
3661 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
3721 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
3781 cgccgccggg atcactctcg gcatggacga gctgtacaag gaattctaac tagagctcgc
3841 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
3901 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
3961 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
4021 aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta
4081 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
4141 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
4201 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
4261 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
4321 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
4381 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
4441 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
4501 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
4561 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
4621 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
4681 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
4741 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
4801 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
4861 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
4921 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
4981 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
5041 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
5101 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
5161 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
5221 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
5281 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
5341 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
5401 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
5461 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
5521 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
5581 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
5641 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
5701 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
5761 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
5821 aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta
5881 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
5941 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
6001 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
6061 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
6121 gagcgtcaga ccccgtagaa aaaatcaaag gatcttcttg agatcctttt tttctgcgcg
6181 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
6241 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
6301 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
6361 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
6421 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
6481 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
6541 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
6601 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
6661 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
6721 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
6781 ccttttgctg gccttttgct ca
//
