LOCUS Exported 4492 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION sgRNA shuttle vector; module 1. ACCESSION . VERSION . KEYWORDS pDGE5 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4492) 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 4492) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4492 /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" promoter 670..772 /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" CDS 773..1432 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(839..858) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 1774..2079 /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 1958..1977 /label=ccdB-fwd /note="ccdB gene, forward primer" misc_RNA 2117..2192 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(2271..2287) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2271..2287) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2284..2306) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2295..2311 /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(2319..2349) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2364..2385 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2502..2519) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2673..3261) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2753..2772) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3432..4292) /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 4055..4074 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4293..4397) /gene="bla" /label=AmpR promoter primer_bind 4465..4483 /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 ttggtctcat tgcgaatatc atccggtgca gcagctgctg cagcaattaa 481 acttgcacag aggccagaaa acgctgggaa gctatttgtg gcgatattcc cgagtttcgg 541 ggagaggtat ctatcaacgg tactaagttg aaaacaatct tcaaaagtcc cacatcgctt 601 agataagaaa acgaagctga gtttatatac agctagagtc gaagtagtga ttgaagtctt 661 ctgagacgtt gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact 721 accgggcgta ttttttgagt tatcgagatt ttcaggagct aaggaagcta aaatggagaa 781 aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga 841 ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc 901 ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct 961 tgcccgcctg atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt 1021 gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc 1081 atcgctctgg agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga 1141 tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt 1201 tttcgtctca gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat 1261 ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt 1321 gctgatgccg ctggcgattc aggttcatca tgccgtctgt gatggcttcc atgtcggcag 1381 aatgcttaat gaattacaac agtactgcga tgagtggcag ggcggggcgt aatcgcgtgg 1441 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat 1501 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag 1561 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca 1621 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac 1681 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg 1741 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa 1801 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg 1861 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa 1921 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc 1981 agtgtgccgg tttccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac 2041 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac 2101 agccagtcga agacttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 2161 atcaacttga aaaagtggca ccgagtcggt gctttttttt cccttgagac caaatcggat 2221 cccgggcccg tcgactgcag aggcctgcat gcaagcttgg cgtaatcatg gtcatagctg 2281 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 2341 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 2401 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 2461 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 2521 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 2581 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 2641 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 2701 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 2761 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 2821 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 2881 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 2941 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 3001 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 3061 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 3121 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 3181 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 3241 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 3301 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 3361 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 3421 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 3481 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 3541 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 3601 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 3661 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 3721 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 3781 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 3841 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 3901 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 3961 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 4021 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 4081 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 4141 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 4201 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 4261 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 4321 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 4381 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 4441 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tc //