LOCUS       sequence-225353-        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"
     primer_bind     250..269
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        484..518
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        539..614
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(join(876..1667,1668..1736))
                     /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     1499..1518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1737..1841)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(1944..2183,1..349)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
       61 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      121 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      181 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      241 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      301 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      361 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      421 gttatcccct gattctgtgg ataaccgtgc ggcccgagag catcgatcct agcatggatg
      481 catttgacag ctagctcagt cctaggtata atgctagccc ataaattgtc aacacgcagt
      541 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      601 caccgagtcg gtgctttttt tccggcttat cggtcagttt cacctgattt acgtaaaaac
      661 ccgcttcggc gggtttttgc ttttggaggg gcagaaagat gaatgactgt ccacgacgct
      721 atacccaaaa gaaaaagctt gaaagaatcg atcgatcccg aatgagtagt acagcggccg
      781 cgattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
      841 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
      901 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
      961 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgggac
     1021 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1081 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1141 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     1201 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     1261 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     1321 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     1381 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     1441 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     1501 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     1561 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     1621 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     1681 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     1741 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     1801 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     1861 ccacctgtca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     1921 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     1981 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2041 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     2101 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2161 ctaatcctgt taccagtggc tgc
//
