LOCUS       sequence-229442-        2183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sgRNA targeting position 6 in pColE1_70a_deGFP_KanR with two
            mismatches in the 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_M2S_AmpR
FEATURES             Location/Qualifiers
     source          1..2183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        31..65
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        86..161
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(join(423..1214,1215..1283))
                     /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     1046..1065
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1284..1388)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1491..2079
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1980..1999
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgagagcat cgatcctagc atggatgcat ttgacagcta gctcagtcct aggtataatg
       61 ctagcggtaa aattgtcaac acggtgtttt agagctagaa atagcaagtt aaaataaggc
      121 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttcc ggcttatcgg
      181 tcagtttcac ctgatttacg taaaaacccg cttcggcggg tttttgcttt tggaggggca
      241 gaaagatgaa tgactgtcca cgacgctata cccaaaagaa aaagcttgaa agaatcgatc
      301 gatcccgaat gagtagtaca gcggccgcga ttatcaaaaa ggatcttcac ctagatcctt
      361 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
      421 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
      481 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
      541 cccagtgctg caatgatacc gcgggaccca cgctcaccgg ctccagattt atcagcaata
      601 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
      661 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
      721 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
      781 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
      841 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
      901 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
      961 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     1021 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     1081 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     1141 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     1201 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     1261 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     1321 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     1381 gttccgcgca catttccccg aaaagtgcca cctgtcatga ccaaaatccc ttaacgtgag
     1441 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     1501 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     1561 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     1621 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     1681 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     1741 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     1801 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1861 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1921 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1981 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     2041 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     2101 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     2161 gattctgtgg ataaccgtgc ggc
//
