LOCUS       sequence-445706-        3524 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  crRNA scaffold for RfxCas13d expressed from hU6 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 3524)
  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 3524)
  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-crScaffold_SV40-BFP
FEATURES             Location/Qualifiers
     source          1..3524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      83..671
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     572..591
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        734..982
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     734..754
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     905..924
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        984..1019
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        1038..1073
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        1141..1438
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1308..1442
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2214..2231)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2220..2444
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2471..2575
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2576..2644,2645..3436)
                     /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(2794..2813)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
       61 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
      121 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
      181 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
      241 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
      301 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
      361 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
      421 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
      481 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
      541 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
      601 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      661 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      721 ttgctcagct agcgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      781 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
      841 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      901 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      961 cttgtggaaa ggacgaaaca ccgcaagtaa acccctacca actggtcggg gtttgaaacg
     1021 ggtcttcgag aagacctcaa gtaaacccct accaactggt cggggtttga aacttttttt
     1081 ttcacctatg tatcgtatca agtactagaa ctccccagtg ggaattcgtg tgtcagttag
     1141 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     1201 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     1261 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     1321 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     1381 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     1441 gctaccagac ccaagcttgg taccgagctc ggatccaccg gtcgccacca tgtccgaact
     1501 gattaaagag aacatgcaca tgaaacttta tatggaaggt acggtcgaca accaccattt
     1561 caaatgtacc tccgaaggag agggcaaacc ctacgagggc acccaaacga tgagaattaa
     1621 agtagtagaa ggtgggccgc tgccctttgc ctttgacatt ctggcaactt cttttctcta
     1681 cgggagtaaa acttttatca accatacgca aggcattcca gacttcttta agcagtcatt
     1741 tcccgagggg tttacttggg agcgggtcac aacatacgag gacggcggcg tgctgaccgc
     1801 tactcaggac accagcctgc aagacgggtg tttgatctac aatgtgaaga ttcgaggcgt
     1861 aaactttact tcaaacggcc cagttatgca gaaaaaaacc ctcggctggg aggcttttac
     1921 agagactctt tacccagccg acggtggttt ggaaggccga aacgacatgg cattgaaatt
     1981 ggttggcggg agccacctta tagcgaatat aaagacgacc tatcggtcta aaaagcccgc
     2041 caaaaatttg aagatgcctg gtgtatatta cgtcgactat aggcttgaga gaataaaaga
     2101 agctaacaac gaaacctatg tggagcaaca tgaggttgca gttgcacgct actgcgatct
     2161 gccgagcaaa ttggggcaca agctgaattg atcagctaga gctcgctaat cagcctcgac
     2221 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2281 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2341 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2401 ggaagataat agcaggcatg ctggggatgc ggtgggctct atggtctaga tttagggtag
     2461 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2521 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2581 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2641 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2701 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2761 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     2821 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2881 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2941 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     3001 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     3061 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     3121 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3181 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3241 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg
     3301 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3361 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3421 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3481 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttg
//
