LOCUS       sequence-445965-        3544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  crRNA scaffold for RfxCas13d expressed from hU6-2xTetO promoter and 
            reporter BFP protein expressed from SV40 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3544)
  AUTHORS   Wan Y, Helenek C, Coraci D, Balazsi G
  TITLE     Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA 
            Downregulation with Minimal Collateral RNA Cutting.
  JOURNAL   ACS Synth Biol. 2024 Oct 18;13(10):3212-3230. doi: 
            10.1021/acssynbio.4c00271. Epub 2024 Oct 8.
  PUBMED    39377757
REFERENCE   2  (bases 1 to 3544)
  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"; doi: 
            "10.1021/acssynbio.4c00271"; journalName: "ACS Synth Biol"; date: 
            "2024-10-18- 18"; volume: "13"; issue: "10"; pages: "3212-3230"
COMMENT     Sequence Label: pUC-hU6-2O-crScaffold_SV40-BFP
FEATURES             Location/Qualifiers
     source          1..3544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      51..639
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     540..559
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     702..722
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     873..892
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    903..921
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        972..1007
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        1026..1061
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        1129..1426
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1296..1430
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2202..2219)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2208..2432
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2459..2563
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2564..2632,2633..3424)
                     /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     complement(2782..2801)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
       61 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
      121 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
      181 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
      241 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
      301 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
      361 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
      421 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
      481 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
      541 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
      601 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
      661 ttacggttcc tggccttttg ctggcctttt gctcagctag cgagggccta tttcccatga
      721 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga
      781 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
      841 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
      901 actccctatc agtgatagag attatatatc tccctatcag tgatagacac cgtggaaagg
      961 acgaaacacc gcaagtaaac ccctaccaac tggtcggggt ttgaaacggg tcttcgagaa
     1021 gacctcaagt aaacccctac caactggtcg gggtttgaaa cttttttttt cacctatgta
     1081 tcgtatcaag tactagaact ccccagtggg aattcgtgtg tcagttaggg tgtggaaagt
     1141 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     1201 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     1261 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     1321 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     1381 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc taccagaccc
     1441 aagcttggta ccgagctcgg atccaccggt cgccaccatg tccgaactga ttaaagagaa
     1501 catgcacatg aaactttata tggaaggtac ggtcgacaac caccatttca aatgtacctc
     1561 cgaaggagag ggcaaaccct acgagggcac ccaaacgatg agaattaaag tagtagaagg
     1621 tgggccgctg ccctttgcct ttgacattct ggcaacttct tttctctacg ggagtaaaac
     1681 ttttatcaac catacgcaag gcattccaga cttctttaag cagtcatttc ccgaggggtt
     1741 tacttgggag cgggtcacaa catacgagga cggcggcgtg ctgaccgcta ctcaggacac
     1801 cagcctgcaa gacgggtgtt tgatctacaa tgtgaagatt cgaggcgtaa actttacttc
     1861 aaacggccca gttatgcaga aaaaaaccct cggctgggag gcttttacag agactcttta
     1921 cccagccgac ggtggtttgg aaggccgaaa cgacatggca ttgaaattgg ttggcgggag
     1981 ccaccttata gcgaatataa agacgaccta tcggtctaaa aagcccgcca aaaatttgaa
     2041 gatgcctggt gtatattacg tcgactatag gcttgagaga ataaaagaag ctaacaacga
     2101 aacctatgtg gagcaacatg aggttgcagt tgcacgctac tgcgatctgc cgagcaaatt
     2161 ggggcacaag ctgaattgat cagctagagc tcgctaatca gcctcgactg tgccttctag
     2221 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2281 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2341 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagataatag
     2401 caggcatgct ggggatgcgg tgggctctat ggtctagatt tagggtaggg gaaatgtgcg
     2461 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2521 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2581 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2641 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2701 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2761 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     2821 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2881 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     2941 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3001 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3061 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3121 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3181 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3241 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     3301 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3361 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3421 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3481 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3541 tgag
//
