LOCUS       Exported                4459 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  M1E shuttle vector containing the Solanum lycopersicum U6 promoter 
            for preparation of single guide RNA transcriptional units by cloning
            of hybridized oligonucleotides..
ACCESSION   .
VERSION     .
KEYWORDS    pDGE654
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4459)
  AUTHORS   Stuttmann J, Barthel K, Martin P, Ordon J, Erickson JL, Herr R, 
            Ferik F, Kretschmer C, Berner T, Keilwagen J, Marillonnet S, Bonas U
  TITLE     Highly efficient multiplex editing: One-shot generation of 8x 
            Nicotiana benthamiana and 12x Arabidopsis mutants.
  JOURNAL   Plant J. 2021 Feb 12. doi: 10.1111/tpj.15197.
  PUBMED    33577114
REFERENCE   2  (bases 1 to 4459)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4459
                     /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        625..727
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             728..1381
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG"
     primer_bind     complement(794..813)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1731..2036
                     /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     1915..1934
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2238..2254)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2238..2254)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2251..2273)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2262..2278
                     /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(2286..2316)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2331..2352
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2469..2486)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2640..3228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2720..2739)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3399..4259)
                     /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     4022..4041
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4260..4364)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4432..4450
                     /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 tgcgttttcc cagtcacgac caagagaaat aagaaaaagg
      481 ccggcccatt tagattgaag atccaggccc aggcccgtaa aagaaaccca acaagcaaat
      541 tctccctcat cgcttataca aagctacttt gcctcgttta tatagcggaa tatgaacatg
      601 tatgattgaa gtcttctgag acgttgatcg gcacgtaaga ggttccaact ttcaccataa
      661 tgaaataaga tcactaccgg gcgtattttt tgagttatcg agattttcag gagctaagga
      721 agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg
      781 taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca
      841 gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc
      901 ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa
      961 agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca
     1021 aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca
     1081 catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt
     1141 tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt
     1201 aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac
     1261 gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg
     1321 cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggggg
     1381 gcgtaaacgc cgcgtggatc cggcttacta aaagccagat aacagtatgc gtatttgcgc
     1441 gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat gtcaaaaaga
     1501 ggtatgctat gaagcagcgt attacagtga cagttgacag cgacagctat cagttgctca
     1561 aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga atgaagcccg
     1621 tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg
     1681 gtttattgaa atgaacggct cttttgctga cgagaacagg ggctggtgaa atgcagttta
     1741 aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata
     1801 ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag
     1861 ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga
     1921 tgaccaccga tatggccagt gtgccggttt ccgttatcgg ggaagaagtg gctgatctca
     1981 gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt
     2041 caggctccct tatacacagc cagtcgaaga cttgtttcag agctatgctg gaaacagcat
     2101 agcaagttga aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     2161 tttttttgct tagagacctt atcggatccc gggcccgtcg actgcagagg cctgcatgca
     2221 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2281 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2341 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2401 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2461 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2521 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2581 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2641 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2701 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2761 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2821 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2881 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2941 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3001 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3061 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3121 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3181 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3241 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3301 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3361 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3421 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3481 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3541 gatccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3601 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3661 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3721 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3781 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3841 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3901 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3961 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4021 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4081 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4141 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4201 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4261 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4321 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4381 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     4441 atcacgaggc cctttcgtc
//
