LOCUS pUC gRNA Shuttle 3142 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Encodes a template from which gRNAs can be made via InFusion
cloning. The Medicago truncatula U6.6 promoter drives the gRNA. For
use in plants..
ACCESSION .
VERSION .
KEYWORDS pUC gRNA Shuttle
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3142)
AUTHORS Jacobs TB, LaFayette PR, Schmitz RJ, Parrott WA.
TITLE Targeted genome modifications in soybean with CRISPR/Cas9.
JOURNAL BMC Biotechnology. 2015;15:16
REFERENCE 2 (bases 1 to 3142)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
//www.snapgene.com
FEATURES Location/Qualifiers
source 1..3142
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(10..28)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 96..200
/gene="bla"
/label=AmpR promoter
CDS 201..1061
/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(419..438)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 1232..1820
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1721..1740
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 1974..1991
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 2108..2129
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2144..2174
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2182..2198
/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 2187..2209
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 2206..2222
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 2206..2222
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
misc_RNA 2640..2715
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
primer_bind complement(2748..2765)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(2748..2764)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2757..2779)
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind complement(2973..2992)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 3092..3114
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
ORIGIN
1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
1621 agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
2221 accatgatta cgccaagctt attaccctgt tatccctaga tatcgatttt ggctacctta
2281 aggtagcatg cctatcttat atgatcaatg aggcatttaa ttgggtgcat atgatggtga
2341 aaaaaggtgc agctcctggc ttgggaatga tgactcatgt ggaatttggt cttaaattta
2401 tcacatcctt ttgggatgtg atgattgtat cacttgttca ttttgcaaag acaaggtgca
2461 ctgctacaaa ctttggttta atctgaaata aaacaaaact cactgagagg aagatgcatc
2521 ccagtaggtg aaagtcgaga aggatttgca tgttactatt acacttgctt tttagtccca
2581 catcgtctga aacataaaat atttcagcgt ttaaatactt caagcgaacc agtaggcttg
2641 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
2701 gcaccgagtc ggtgcttttt ttgctacctt aagagagtca tgaattcact ggccgtcgtt
2761 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
2821 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
2881 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc
2941 ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
3001 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
3061 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
3121 ccgtcatcac cgaaacgcgc ga
//
