LOCUS       sequence-307146-        4449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M6 shuttle vector containing the Solanum lycopersicum U3 promoter 
            for preparation of single guide RNA transcriptional units by cloning
            of hybridized oligonucleotides..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4449)
  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 4449)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant J. 
            2021 Feb 12. doi: 10.1111/tpj.15197."
COMMENT     Sequence Label: pDGE499
FEATURES             Location/Qualifiers
     source          1..4449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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        617..719
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             720..1403
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(786..805)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1723..2028
                     /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     1907..1926
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2228..2244)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2228..2244)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2241..2263)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2252..2268
                     /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(join(2276..2282,2283..2300,2301..2306))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2321..2342
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2459..2476)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2630..3218)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2710..2729)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3389..4180,4181..4249))
                     /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     4012..4031
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4250..4354)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4422..4440
                     /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 gcgcaaaaac ttcaaaatct aacacaaatt tttattgtat
      481 ttgagcagat ttgcataata gtcagcgaag ctgtgtcaag ggccaacata ttattcccac
      541 atcggtaggt caacacataa actccaggtt tatgaggctt cgtagcagta gcacatgtca
      601 aagtcttctg agacgttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa
      661 gatcactacc gggcgtattt tttgagttat cgagattttc aggagctaag gaagctaaaa
      721 tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac
      781 attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata
      841 ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc
      901 acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg
      961 agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa
     1021 cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt
     1081 cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga
     1141 atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg
     1201 ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg
     1261 acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg
     1321 tcggtagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggg gggcgtaaac
     1381 gccgcgtgga tccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt
     1441 ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct
     1501 atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat
     1561 atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc
     1621 gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg
     1681 aaatgaacgg ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac
     1741 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
     1801 acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
     1861 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
     1921 gatatggcca gtgtgccggt ttccgttatc ggggaagaag tggctgatct cagccaccgc
     1981 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc
     2041 cttatacaca gccagtcgaa gacttgtttc agagctatgc tggaaacagc atagcaagtt
     2101 gaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttttc
     2161 cagtgagacc atcggatccc gggcccgtcg actgcagagg cctgcatgca agcttggcgt
     2221 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2281 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2341 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2401 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2461 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2521 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2581 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2641 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2701 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2761 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2821 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2881 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2941 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3001 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3061 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3121 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3181 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3241 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3301 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3361 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3421 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3481 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gatccacgct
     3541 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3601 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3661 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3721 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3781 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3841 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3901 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3961 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4021 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4081 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4141 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4201 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4261 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4321 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4381 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     4441 cctttcgtc
//
