LOCUS Exported 4371 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION sgRNA shuttle vector; module 7. ACCESSION . VERSION . KEYWORDS pDGE14 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4371) 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 4371) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4371 /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(2150..2166) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2150..2166) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2163..2185) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2174..2190 /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(2198..2228) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2243..2264 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2381..2398) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2552..3140) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2632..2651) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3311..4171) /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 3934..3953 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4172..4276) /gene="bla" /label=AmpR promoter primer_bind 4344..4362 /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 ttggtctcac cagtaagttg 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 cgaatgagac caatcggatc 2101 ccgggcccgt cgactgcaga ggcctgcatg caagcttggc gtaatcatgg tcatagctgt 2161 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 2221 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 2281 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 2341 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 2401 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 2461 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 2521 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 2581 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 2641 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 2701 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 2761 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 2821 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 2881 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 2941 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 3001 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 3061 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 3121 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 3181 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 3241 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 3301 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 3361 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 3421 catctggccc cagtgctgca atgataccgc gagaaccacg ctcaccggct ccagatttat 3481 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 3541 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 3601 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 3661 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 3721 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 3781 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 3841 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 3901 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 3961 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 4021 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 4081 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 4141 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 4201 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 4261 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 4321 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c //