LOCUS Exported 4372 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION sgRNA shuttle vector; module 6. ACCESSION . VERSION . KEYWORDS pDGE13 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4372) 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 4372) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4372 /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 550..652 /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" CDS 653..1312 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(719..738) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 1654..1959 /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 1838..1857 /label=ccdB-fwd /note="ccdB gene, forward primer" misc_RNA 1997..2072 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(2151..2167) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2151..2167) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2164..2186) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2175..2191 /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(2199..2229) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2244..2265 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2382..2399) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2553..3141) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2633..2652) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3312..4172) /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 3935..3954 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4173..4277) /gene="bla" /label=AmpR promoter primer_bind 4345..4363 /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 ccgtaagttg aaaacaatct tcaaaagtcc cacatcgctt 481 agataagaaa acgaagctga gtttatatac agctagagtc gaagtagtga ttgaagtctt 541 ctgagacgtt gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact 601 accgggcgta ttttttgagt tatcgagatt ttcaggagct aaggaagcta aaatggagaa 661 aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga 721 ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc 781 ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct 841 tgcccgcctg atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt 901 gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc 961 atcgctctgg agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga 1021 tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt 1081 tttcgtctca gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat 1141 ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt 1201 gctgatgccg ctggcgattc aggttcatca tgccgtctgt gatggcttcc atgtcggcag 1261 aatgcttaat gaattacaac agtactgcga tgagtggcag ggcggggcgt aatcgcgtgg 1321 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat 1381 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag 1441 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca 1501 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac 1561 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg 1621 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa 1681 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg 1741 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa 1801 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc 1861 agtgtgccgg tttccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac 1921 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac 1981 agccagtcga agacttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 2041 atcaacttga aaaagtggca ccgagtcggt gctttttttt ccagtgagac caaatcggat 2101 cccgggcccg tcgactgcag aggcctgcat gcaagcttgg cgtaatcatg gtcatagctg 2161 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 2221 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 2281 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 2341 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 2401 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 2461 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 2521 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 2581 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 2641 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 2701 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 2761 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 2821 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 2881 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 2941 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 3001 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 3061 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 3121 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 3181 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 3241 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 3301 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 3361 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 3421 ccatctggcc ccagtgctgc aatgataccg cgagaaccac gctcaccggc tccagattta 3481 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 3541 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 3601 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 3661 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 3721 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 3781 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 3841 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 3901 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 3961 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 4021 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 4081 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 4141 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 4201 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 4261 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 4321 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tc //