LOCUS       sequence-305800-        4386 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M4 shuttle vector containing the Arabidopsis thaliana 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 4386)
  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 4386)
  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: pDGE336
FEATURES             Location/Qualifiers
     source          1..4386
                     /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        551..653
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             654..1337
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(720..739)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1657..1962
                     /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     1841..1860
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2165..2181)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2165..2181)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2178..2200)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2189..2205
                     /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(2213..2219,2220..2237,2238..2243))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2258..2279
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2396..2413)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2567..3155)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2647..2666)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3326..4117,4118..4186))
                     /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     3949..3968
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4187..4291)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4359..4377
                     /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 atggtaagtt gaaaacaatc ttcaaaagtc ccacatcgct
      481 tagataagaa aacgaagctg agtttatata cagctagagt cgaagtagtg attgaagtct
      541 tctgagacgt tgatcggcac gtaagaggtt ccaactttca ccataatgaa ataagatcac
      601 taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct aaaatggaga
      661 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
      721 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
      781 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
      841 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
      901 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
      961 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     1021 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     1081 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     1141 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     1201 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     1261 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggggggcgt aaacgccgcg
     1321 tggatccggc ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg atttttgcgg
     1381 tataagaata tatactgata tgtatacccg aagtatgtca aaaagaggta tgctatgaag
     1441 cagcgtatta cagtgacagt tgacagcgac agctatcagt tgctcaaggc atatatgatg
     1501 tcaatatctc cggtctggta agcacaacca tgcagaatga agcccgtcgt ctgcgtgccg
     1561 aacgctggaa agcggaaaat caggaaggga tggctgaggt cgcccggttt attgaaatga
     1621 acggctcttt tgctgacgag aacaggggct ggtgaaatgc agtttaaggt ttacacctat
     1681 aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccc
     1741 gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt
     1801 gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg
     1861 gccagtgtgc cggtttccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat
     1921 gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg ctcccttata
     1981 cacagccagt cgaagacttg tttcagagct atgctggaaa cagcatagca agttgaaata
     2041 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tttgacttga
     2101 gaccaaaatc ggatcccggg cccgtcgact gcagaggcct gcatgcaagc ttggcgtaat
     2161 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     2221 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     2281 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     2341 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     2401 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2461 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     2521 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     2581 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2641 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2701 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     2761 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     2821 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     2881 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     2941 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3001 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3061 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3121 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3181 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3241 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3301 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3361 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3421 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagat ccacgctcac
     3481 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3541 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3601 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3661 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3721 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     3781 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     3841 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     3901 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     3961 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4021 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4081 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4141 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4201 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4261 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     4321 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     4381 ttcgtc
//
