LOCUS       sequence-229447-        2183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sgRNA targeting position 6 in pColE1_70a_deGFP_KanR with 
            seven 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_M7NS_AmpR
FEATURES             Location/Qualifiers
     source          1..2183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        50..84
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        105..180
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(join(442..1233,1234..1302))
                     /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     1065..1084
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1303..1407)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1510..2098
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1999..2018
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgtggataa ccgtgcggcc cgagagcatc gatcctagca tggatgcatt tgacagctag
       61 ctcagtccta ggtataatgc tagcccattt tttgtcaaca cgcagtttta gagctagaaa
      121 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
      181 tttttttccg gcttatcggt cagtttcacc tgatttacgt aaaaacccgc ttcggcgggt
      241 ttttgctttt ggaggggcag aaagatgaat gactgtccac gacgctatac ccaaaagaaa
      301 aagcttgaaa gaatcgatcg atcccgaatg agtagtacag cggccgcgat tatcaaaaag
      361 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
      421 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
      481 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
      541 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgggacccac gctcaccggc
      601 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
      661 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
      721 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
      781 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
      841 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
      901 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
      961 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     1021 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     1081 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     1141 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     1201 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     1261 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     1321 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1381 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgtcatgac
     1441 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1501 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1561 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1621 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     1681 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1741 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1801 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1861 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1921 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1981 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2041 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     2101 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2161 ctttcctgcg ttatcccctg att
//
