LOCUS       sequence-227542-        1778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro expression of anti-mRFP1 Cas9 sgRNA from the T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 1778)
  AUTHORS   Stark JC, Huang A, Hsu KJ, Dubner RS, Forbrook J, Marshalla S, 
            Rodriguez F, Washington M, Rybnicky GA, Nguyen PQ, Hasselbacher B, 
            Jabri R, Kamran R, Koralewski V, Wightkin W, Martinez T, Jewett MC
  TITLE     BioBits Health: Classroom Activities Exploring Engineering, Biology,
            and Human Health with Fluorescent Readouts.
  JOURNAL   ACS Synth Biol. 2019 May 7. doi: 10.1021/acssynbio.8b00381.
  PUBMED    30925042
REFERENCE   2  (bases 1 to 1778)
  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: "ACS Synth 
            Biol. 2019 May 7. doi: 10.1021/acssynbio.8b00381."
COMMENT     Sequence Label: pJL1-mRFP1 gRNA
FEATURES             Location/Qualifiers
     source          1..1778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      111..699
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     600..619
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        727..745
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     ncRNA           752..788
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        809..884
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(952..1767)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1675..1694
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1759..1778
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg
       61 caatgtaaca tcagagattt tgagacacaa cgtgagatca aaggatcttc ttgagatcct
      121 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
      181 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
      241 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
      301 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
      361 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
      421 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
      481 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
      541 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
      601 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
      661 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcgatcccg
      721 cgaaattaat acgactcact ataggcgcat gctgatgagt ccgtgaggac gaaacgagta
      781 agctcgtcca tgcgtttcaa agttcgtagt tttagagcta gaaatagcaa gttaaaataa
      841 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tataacccct
      901 tggggcctct aaacgggtct tgaggggttt tttgctgaaa gccaattctg attagaaaaa
      961 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     1021 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     1081 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     1141 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     1201 tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     1261 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     1321 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     1381 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     1441 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     1501 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     1561 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     1621 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     1681 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcttcgagca
     1741 agacgtttcc cgttgaatat ggctcataac accccttg
//
