LOCUS       sequence-273629-        2479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Subcloning and expression of mCherry-targeting sgRNA M for use in 
            Traffic Light reporter system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2479)
  AUTHORS   Standage-Beier K, Brookhouser N, Balachandran P, Zhang Q, Brafman 
            DA, Wang X
  TITLE     RNA-Guided Recombinase-Cas9 Fusion Targets Genomic DNA Deletion and 
            Integration.
  JOURNAL   CRISPR J. 2019 Aug;2:209-222. doi: 10.1089/crispr.2019.0013.
  PUBMED    31436506
REFERENCE   2  (bases 1 to 2479)
  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: "CRISPR J.";
            date: "2019-08"; volume: "2:209-222. doi"; pages: " 
            10.1089/crispr.2019.0013"
COMMENT     Sequence Label: pSB1C3-hsg(M)
FEATURES             Location/Qualifiers
     source          1..2479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     96..114
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(118..161)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     promoter        170..418
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     170..190
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     341..360
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     regulatory      427..436
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        440..515
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      616..673
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(697..714)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(868..1456)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(948..967)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1639..1733)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1754..2413)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2328..2347
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 attatggtga aagttggaac ctcttacgtg cccgatcaac tcgagtgcca cctgacgtct
       61 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggcagaatt
      121 tcagataaaa aaaatcctta gctttcgcta aggatgattt ctggaattcg agggcctatt
      181 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
      241 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
      301 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
      361 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgt
      421 tgctcaccat ggtggcgacg ttttagagct agaaatagca agttaaaata aggctagtcc
      481 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag ctagaaatag
      541 caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag acaaatctag
      601 tagcggccgc tgcagtccgg caaaaaaggg caaggtgtca ccaccctgcc ctttttcttt
      661 aaaaccgaaa agattacttc gcgttatgca ggcttcctcg ctcactgact cgctgcgctc
      721 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
      781 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
      841 ccgtaaaaag gccgcgttgc tggcgttttt ccacaggctc cgcccccctg acgagcatca
      901 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
      961 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     1021 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     1081 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     1141 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     1201 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     1261 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     1321 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     1381 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     1441 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     1501 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     1561 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     1621 tgacagctcg aggcttggat tctcaccaat aaaaaacgcc cggcggcaac cgagcgttct
     1681 gaacaaatcc agatggagtt ctgaggtcat tactggatct atcaacagga gtccaagcga
     1741 gctcgatatc aaattacgcc ccgccctgcc actcatcgca gtactgttgt aattcattaa
     1801 gcattctgcc gacatggaag ccatcacaaa cggcatgatg aacctgaatc gccagcggca
     1861 tcagcacctt gtcgccttgc gtataatatt tgcccatggt gaaaacgggg gcgaagaagt
     1921 tgtccatatt ggccacgttt aaatcaaaac tggtgaaact cacccaggga ttggctgaga
     1981 cgaaaaacat attctcaata aaccctttag ggaaataggc caggttttca ccgtaacacg
     2041 ccacatcttg cgaatatatg tgtagaaact gccggaaatc gtcgtggtat tcactccaga
     2101 gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta acaagggtga acactatccc
     2161 atatcaccag ctcaccgtct ttcattgcca tacgaaattc cggatgagca ttcatcaggc
     2221 gggcaagaat gtgaataaag gccggataaa acttgtgctt atttttcttt acggtcttta
     2281 aaaaggccgt aatatccagc tgaacggtct ggttataggt acattgagca actgactgaa
     2341 atgcctcaaa atgttcttta cgatgccatt gggatatatc aacggtggta tatccagtga
     2401 tttttttctc cattttagct tccttagctc ctgaaaatct cgataactca aaaaatacgc
     2461 ccggtagtga tcttatttc
//
