LOCUS       sequence-229445-        2183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sgRNA targeting position 6 in pColE1_70a_deGFP_KanR with 
            four mismatches 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_M4NS_AmpR
FEATURES             Location/Qualifiers
     source          1..2183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..106
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        127..202
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(join(464..1255,1256..1324))
                     /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     1087..1106
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1325..1429)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1532..2120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2021..2040
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttcctgcgt tatcccctga ttctgtggat aaccgtgcgg cccgagagca tcgatcctag
       61 catggatgca tttgacagct agctcagtcc taggtataat gctagcccat aaattgtcaa
      121 cacgcagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      181 aaaagtggca ccgagtcggt gctttttttc cggcttatcg gtcagtttca cctgatttac
      241 gtaaaaaccc gcttcggcgg gtttttgctt ttggaggggc agaaagatga atgactgtcc
      301 acgacgctat acccaaaaga aaaagcttga aagaatcgat cgatcccgaa tgagtagtac
      361 agcggccgcg attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
      421 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
      481 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
      541 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
      601 cgcgggaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
      661 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
      721 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
      781 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
      841 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
      901 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
      961 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     1021 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     1081 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     1141 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     1201 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     1261 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     1321 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     1381 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     1441 gaaaagtgcc acctgtcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1501 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1561 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1621 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1681 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1741 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1801 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1861 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1921 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1981 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2041 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     2101 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     2161 gctggccttt tgctcacatg ttc
//
