LOCUS       sequence-419127-        3346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of sgRNAs for expression in plants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3346)
  TITLE     Christian Staehelin Lab Plasmids 2023
REFERENCE   2  (bases 1 to 3346)
  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"
COMMENT     Sequence Label: pUC-tracrRNA-PMtU6-1
FEATURES             Location/Qualifiers
     source          1..3346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        20..95
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(700..717)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(700..716)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(709..731)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(925..944)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1044..1066
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1104..1122)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1190..1294
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1295..1363,1364..2155)
                     /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     complement(1513..1532)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2326..2914
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2815..2834
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3068..3085
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3202..3223
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3238..3243,3244..3261,3262..3268)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3276..3292
                     /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)."
     primer_bind     3281..3303
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3300..3316
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3300..3316
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ggatccagcg tgggtctcgg ttttagagct agaaatagca agttaaaata aggctagtcc
       61 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttcaagagct tggagtggat
      121 ggaattttcc tccgttttac ctgtggaatc ggcagcaaag gaatccaaca tttcacttga
      181 gttaactcaa tagcaagaat aacgtccata gtttcagcat tcaagcaaaa cagccaagaa
      241 aatcagcttg gtaatttcag tgagacttgg actaccataa gcagcaccgc ctattacact
      301 taatggggta aagtaaaacg agccacatca cctccttgat tttaaggagc atttgaagga
      361 gtataaaaag aatgtatgta atgtaaggtt gtgttgtgtc attcaagata gcaagacgga
      421 ccaaagcttc tatgtatcta tctatgtcta tgatatgatg attgtattga tttggtttga
      481 gtacagtgag ggagagggag gaacttcttc acttgtttat ttaacctgaa actcaactca
      541 aatcactgag agtgaatgtt gagaaataag tattatgtta tgtttgcttt gctattagtc
      601 ccacatcgct tacatatacg tcatttatat tgtttatata gcctagacga acagcagggt
      661 ttattgagag acctctgaag ataacatact aagcttggca ctggccgtcg ttttacaacg
      721 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
      781 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
      841 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
      901 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
      961 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     1021 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     1081 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     1141 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1201 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1261 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     1321 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1381 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1441 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     1501 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     1561 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     1621 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     1681 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     1741 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     1801 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     1861 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     1921 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     1981 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2041 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2101 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2161 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     2221 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     2281 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2341 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2401 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2461 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2521 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2581 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2641 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2701 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2761 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2821 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2881 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2941 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3001 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3061 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     3121 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     3181 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     3241 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     3301 aggaaacagc tatgaccatg attacgaatt cgagctcggt acccgg
//
