LOCUS       sequence-229443-        2183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sgRNA targeting position 6 in pColE1_70a_deGFP_KanR with one
            mismatch in the non-seed region.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2183)
  AUTHORS   Collias D, Marshall R, Collins SP, Beisel CL, Noireaux V
  TITLE     An educational module to explore CRISPR technologies with a 
            cell-free transcription-translation system.
  JOURNAL   Synth Biol (Oxf). 2019 Jan 21;4(1):ysz005. doi: 
            10.1093/synbio/ysz005. eCollection 2019.
  PUBMED    32995532
REFERENCE   2  (bases 1 to 2183)
  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: "Synth Biol 
            (Oxf)."; date: "2019-01-21"; volume: "4(1):ysz005. doi"; pages: " 
            10.1093/synbio/ysz005. eCollection 2019"
COMMENT     Sequence Label: pColE1_sgRNA_M1NS_AmpR
FEATURES             Location/Qualifiers
     source          1..2183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        137..171
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        192..267
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(join(529..1320,1321..1389))
                     /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     1152..1171
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1390..1494)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(1597..2183,1..2)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2086..2105
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
       61 tgttctttcc tgcgttatcc cctgattctg tggataaccg tgcggcccga gagcatcgat
      121 cctagcatgg atgcatttga cagctagctc agtcctaggt ataatgctag ccgtaaaatt
      181 gtcaacacgc agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
      241 cttgaaaaag tggcaccgag tcggtgcttt ttttccggct tatcggtcag tttcacctga
      301 tttacgtaaa aacccgcttc ggcgggtttt tgcttttgga ggggcagaaa gatgaatgac
      361 tgtccacgac gctataccca aaagaaaaag cttgaaagaa tcgatcgatc ccgaatgagt
      421 agtacagcgg ccgcgattat caaaaaggat cttcacctag atccttttaa attaaaaatg
      481 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
      541 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
      601 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
      661 gataccgcgg gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
      721 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
      781 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
      841 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
      901 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
      961 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     1021 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     1081 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     1141 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     1201 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     1261 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     1321 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     1381 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     1441 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     1501 tccccgaaaa gtgccacctg tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     1561 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     1621 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     1681 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     1741 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     1801 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     1861 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     1921 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     1981 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     2041 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     2101 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     2161 gtcagggggg cggagcctat gga
//
