LOCUS pBlu/gRNA 3514 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Serves as a shuttle vector for target oligo before insertion into
Cas9 destination vector.
ACCESSION .
VERSION .
KEYWORDS pBlu/gRNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3514)
AUTHORS Michno JM, Wang X, Liu J, Curtin SJ, Kono TJ, Stupar RM
TITLE CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a
new web-tool and a modified Cas9 enzyme.
JOURNAL GM Crops Food. 2015;6(4):243-52. doi: 10.1080/21645698.2015.1106063.
PUBMED 26479970
REFERENCE 2 (bases 1 to 3514)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
//www.snapgene.com
FEATURES Location/Qualifiers
source 1..3514
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 21..125
/gene="bla"
/label=AmpR promoter
CDS 126..986
/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(344..363)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 1157..1745
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1646..1665
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 1899..1916
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 2033..2054
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2069..2099
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2107..2123
/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 2112..2134
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 2131..2147
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 2131..2147
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
promoter 2168..2186
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind 2214..2230
/label=pBluescriptKS
/note="For pBluescript vector"
primer_bind 2215..2231
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
misc_RNA 2719..2794
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
primer_bind complement(2822..2838)
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2822..2838)
/label=pBluescriptSK
/note="For pBluescript vector"
primer_bind complement(2870..2889)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(2871..2889)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2896..2913)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(2896..2912)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2905..2927)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 3054..3509
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3141..3160)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3351..3372
/label=F1ori-F
/note="F1 origin, forward primer"
ORIGIN
1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
1081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
1801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
1861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
1921 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
1981 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
2041 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
2101 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
2161 agctcgaaat taaccctcac taaagggaac aaaagctggt accgggcccc ccctcgaggt
2221 cgacggtatc gataagcttg atatcgaatt caagctttcg ttgaacaacg gaaactcgac
2281 ttgccttccg cacaatacat catttcttct tagctttttt tcttcttctt cgttcataca
2341 gttttttttt gtttatcagc ttacattttc ttgaaccgta gctttcgttt tcttcttttt
2401 aactttccat tcggagtttt tgtatcttgt ttcatagttt gtcccaggat tagaatgatt
2461 aggcatcgaa ccttcaagaa tttgattgaa taaaacatct tcattcttaa gatatgaaga
2521 taatcttcaa aaggcccctg ggaatctgaa agaagagaag caggcccatt tatatgggaa
2581 agaacaatag tatttcttat ataggcccat ttaagttgaa aacaatcttc aaaagtccca
2641 catcgcttag ataagaaaac gaagctgagt ttatatacag ctagagtcga agtagtgatt
2701 gggtcttcga gaagacctgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
2761 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ttgaattcct gcagcccggg
2821 ggatccacta gttctagagc ggccgccacc gcggtggagc tccaattcgc cctatagtga
2881 gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
2941 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
3001 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
3061 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
3121 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
3181 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
3241 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
3301 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
3361 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
3421 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
3481 ttttaacaaa atattaacgc ttacaattta ggtg
//
