LOCUS       sequence-307092-        4454 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M2E 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 4454)
  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 4454)
  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: pDGE476
FEATURES             Location/Qualifiers
     source          1..4454
                     /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        619..721
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             722..1405
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(788..807)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1725..2030
                     /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     1909..1928
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2233..2249)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2233..2249)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2246..2268)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2257..2273
                     /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(2281..2287,2288..2305,2306..2311))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2326..2347
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2464..2481)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2635..3223)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2715..2734)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3394..4185,4186..4254))
                     /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     4017..4036
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4255..4359)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4427..4445
                     /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 ttttggtctc accctcaaaa acttcaaaat ctaacacaaa tttttattgt
      481 atttgagcag atttgcataa tagtcagcga agctgtgtca agggccaaca tattattccc
      541 acatcggtag gtcaacacat aaactccagg tttatgaggc ttcgtagcag tagcacatgt
      601 caaagtcttc tgagacgttg atcggcacgt aagaggttcc aactttcacc ataatgaaat
      661 aagatcacta ccgggcgtat tttttgagtt atcgagattt tcaggagcta aggaagctaa
      721 aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga
      781 acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga
      841 tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat
      901 tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa tgaaagacgg
      961 tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga
     1021 aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata
     1081 ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga
     1141 gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt
     1201 ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg
     1261 cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca
     1321 tgtcggtaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gggggcgtaa
     1381 acgccgcgtg gatccggctt actaaaagcc agataacagt atgcgtattt gcgcgctgat
     1441 ttttgcggta taagaatata tactgatatg tatacccgaa gtatgtcaaa aagaggtatg
     1501 ctatgaagca gcgtattaca gtgacagttg acagcgacag ctatcagttg ctcaaggcat
     1561 atatgatgtc aatatctccg gtctggtaag cacaaccatg cagaatgaag cccgtcgtct
     1621 gcgtgccgaa cgctggaaag cggaaaatca ggaagggatg gctgaggtcg cccggtttat
     1681 tgaaatgaac ggctcttttg ctgacgagaa caggggctgg tgaaatgcag tttaaggttt
     1741 acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg
     1801 acacgcccgg gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag
     1861 tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca
     1921 ccgatatggc cagtgtgccg gtttccgtta tcggggaaga agtggctgat ctcagccacc
     1981 gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa atgtcaggct
     2041 cccttataca cagccagtcg aagacttgtt tcagagctat gctggaaaca gcatagcaag
     2101 ttgaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
     2161 tgcttagaga cctttatcgg atcccgggcc cgtcgactgc agaggcctgc atgcaagctt
     2221 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     2281 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     2341 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     2401 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     2461 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     2521 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     2581 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     2641 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     2701 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     2761 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2821 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2881 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2941 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3001 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3061 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3121 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3181 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3241 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3301 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3361 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3421 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3481 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagatcc
     3541 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3601 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3661 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3721 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3781 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3841 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3901 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3961 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4021 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4081 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4141 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4201 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4261 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4321 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     4381 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     4441 gaggcccttt cgtc
//
