LOCUS       sequence-305915-        4444 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M3 shuttle vector containing the Solanum lycopersicum U6 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 4444)
  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 4444)
  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: pDGE658
FEATURES             Location/Qualifiers
     source          1..4444
                     /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        609..711
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             712..1395
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(778..797)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1715..2020
                     /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     1899..1918
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2223..2239)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2223..2239)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2236..2258)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2247..2263
                     /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(2271..2277,2278..2295,2296..2301))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2316..2337
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2454..2471)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2625..3213)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2705..2724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3384..4175,4176..4244))
                     /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     4007..4026
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4245..4349)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4417..4435
                     /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 tttggtctca cggtcaagag aaataagaaa aaggccggcc catttagatt
      481 gaagatccag gcccaggccc gtaaaagaaa cccaacaagc aaattctccc tcatcgctta
      541 tacaaagcta ctttgcctcg tttatatagc ggaatatgaa catgtatgat tgaagtcttc
      601 tgagacgttg atcggcacgt aagaggttcc aactttcacc ataatgaaat aagatcacta
      661 ccgggcgtat tttttgagtt atcgagattt tcaggagcta aggaagctaa aatggagaaa
      721 aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag
      781 gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc
      841 tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt
      901 gcccgcctga tgaatgctca tccggaattc cgtatggcaa tgaaagacgg tgagctggtg
      961 atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca
     1021 tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat
     1081 gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt
     1141 ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg
     1201 gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg
     1261 ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca tgtcggcaga
     1321 atgcttaatg aattacaaca gtactgcgat gagtggcagg gggggcgtaa acgccgcgtg
     1381 gatccggctt actaaaagcc agataacagt atgcgtattt gcgcgctgat ttttgcggta
     1441 taagaatata tactgatatg tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca
     1501 gcgtattaca gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc
     1561 aatatctccg gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa
     1621 cgctggaaag cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac
     1681 ggctcttttg ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa
     1741 aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg
     1801 gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga
     1861 actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc
     1921 cagtgtgccg gtttccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga
     1981 catcaaaaac gccattaacc tgatgttctg gggaatataa atgtcaggct cccttataca
     2041 cagccagtcg aagacttgtt tcagagctat gctggaaaca gcatagcaag ttgaaataag
     2101 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt tatggtgaga
     2161 ccaaaatcgg atcccgggcc cgtcgactgc agaggcctgc atgcaagctt ggcgtaatca
     2221 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     2281 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     2341 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     2401 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2461 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2521 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2581 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2641 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2701 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2761 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2821 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2881 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2941 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3001 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3061 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3121 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3181 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3241 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3301 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3361 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3421 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3481 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagatcc acgctcaccg
     3541 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3601 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3661 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3721 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3781 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3841 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3901 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3961 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4021 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4081 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4141 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4201 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4261 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4321 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     4381 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     4441 cgtc
//
