LOCUS Exported 4374 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION sgRNA shuttle vector; module 8E. ACCESSION . VERSION . KEYWORDS pDGE15 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4374) AUTHORS Ordon J, Gantner J, Kemna J, Schwalgun L, Reschke M, Streubel J, Boch J, Stuttmann J TITLE Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly genome editing toolkit. JOURNAL Plant J. 2016 Aug 31. doi: 10.1111/tpj.13319. PUBMED 27579989 REFERENCE 2 (bases 1 to 4374) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4374 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 151..170 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 364..386 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 378..395 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 379..395 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" misc_RNA complement(453..528) /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" CDS complement(566..871) /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind complement(668..687) /label=ccdB-fwd /note="ccdB gene, forward primer" CDS complement(1213..1872) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 1787..1806 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(1873..1975) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" primer_bind complement(2153..2169) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2153..2169) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2166..2188) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2177..2193 /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)." promoter complement(2201..2231) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2246..2267 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2384..2401) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2555..3143) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2635..2654) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3314..4174) /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 3937..3956 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4175..4279) /gene="bla" /label=AmpR promoter primer_bind 4347..4365 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 421 tgcatctaga taaggtctct aagcaaaaaa aagcaccgac tcggtgccac tttttcaagt 481 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaacaa gtcttcgact 541 ggctgtgtat aagggagcct gacatttata ttccccagaa catcaggtta atggcgtttt 601 tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc cccgataacg gaaaccggca 661 cactggccat atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt 721 aaagttcacg ggagacttta tctgacagca gacgtgcact ggccaggggg atcaccatcc 781 gtcgcccggg cgtgtcaata atatcactct gtacatccac aaacagacga taacggctct 841 ctcttttata ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc gtcagcaaaa 901 gagccgttca tttcaataaa ccgggcgacc tcagccatcc cttcctgatt ttccgctttc 961 cagcgttcgg cacgcagacg acgggcttca ttctgcatgg ttgtgcttac cagaccggag 1021 atattgacat catatatgcc ttgagcaact gatagctgtc gctgtcaact gtcactgtaa 1081 tacgctgctt catagcatac ctctttttga catacttcgg gtatacatat cagtatatat 1141 tcttataccg caaaaatcag cgcgcaaata cgcatactgt tatctggctt ttagtaagcc 1201 ggatccacgc gattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag 1261 cattctgccg acatggaagc catcacagac ggcatgatga acctgaatcg ccagcggcat 1321 cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt 1381 gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac 1441 gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc 1501 cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag 1561 cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca 1621 tatcaccagc tcaccgtctt tcattgccat acggaattcc ggatgagcat tcatcaggcg 1681 ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa 1741 aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa 1801 tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat 1861 ttttttctcc attttagctt ccttagctcc tgaaaatctc gataactcaa aaaatacgcc 1921 cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc 1981 tcagaagact tcaatcacta cttcgactct agctgtatat aaactcagct tcgttttctt 2041 atctaagcga tgtgggactt ttgaagattg ttttcaactt attcgtgaga ccaaaatcgg 2101 atcccgggcc cgtcgactgc agaggcctgc atgcaagctt ggcgtaatca tggtcatagc 2161 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 2221 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 2281 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 2341 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 2401 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 2461 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 2521 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 2581 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 2641 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 2701 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 2761 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 2821 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 2881 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 2941 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 3001 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 3061 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 3121 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 3181 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 3241 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 3301 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 3361 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 3421 taccatctgg ccccagtgct gcaatgatac cgcgagaacc acgctcaccg gctccagatt 3481 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 3541 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 3601 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 3661 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 3721 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 3781 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 3841 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 3901 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 3961 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 4021 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 4081 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 4141 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 4201 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 4261 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca 4321 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc //