LOCUS       sequence-445705-        4479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SARS-COV-2 targeting crRNA array for RfxCas13d expressed from single
            hU6 promoter and reporter BFP protein expressed from EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4479)
  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 4479)
  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-crCoV-Mutiplex_EF1a-BFP
FEATURES             Location/Qualifiers
     source          1..4479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..255
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7..27
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     178..197
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        257..292
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        315..350
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        373..408
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        431..466
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        521..1699
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          752..1690
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1647..1667
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(2442..2459)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2448..2672
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2699..2803
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2804..2872,2873..3664)
                     /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(3022..3041)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3835..4423
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4324..4343
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctagcgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
       61 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      121 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      181 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      241 aaaggacgaa acaccgcaag taaaccccta ccaactggtc ggggtttgaa accactatta
      301 gcataagcag ttgtcaagta aacccctacc aactggtcgg ggtttgaaac ttgaatctga
      361 gggtccacca aacaagtaaa cccctaccaa ctggtcgggg tttgaaacaa cgccttgtcc
      421 tcgagggaat caagtaaacc cctaccaact ggtcggggtt tgaaactttt tttttcacct
      481 atgtatcgta tcaagtacta gaactcccca gtgggaattc ggctccggtg cccgtcagtg
      541 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
      601 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
      661 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
      721 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc
      781 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta
      841 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc
      901 ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg
      961 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat
     1021 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc
     1081 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg
     1141 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg
     1201 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat
     1261 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg
     1321 gccgcttccc ggccctgctg cagggagctc aaaatgaagg acgcggcgct cgggagagcg
     1381 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg
     1441 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag
     1501 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg
     1561 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct
     1621 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct
     1681 tccatttcag gtgtcgtgac gtacggccac catgagcatg tccgaactga ttaaagagaa
     1741 catgcacatg aaactttata tggaaggtac ggtcgacaac caccatttca aatgtacctc
     1801 cgaaggagag ggcaaaccct acgagggcac ccaaacgatg agaattaaag tagtagaagg
     1861 tgggccgctg ccctttgcct ttgacattct ggcaacttct tttctctacg ggagtaaaac
     1921 ttttatcaac catacgcaag gcattccaga cttctttaag cagtcatttc ccgaggggtt
     1981 tacttgggag cgggtcacaa catacgagga cggcggcgtg ctgaccgcta ctcaggacac
     2041 cagcctgcaa gacgggtgtt tgatctacaa tgtgaagatt cgaggcgtaa actttacttc
     2101 aaacggccca gttatgcaga aaaaaaccct cggctgggag gcttttacag agactcttta
     2161 cccagccgac ggtggtttgg aaggccgaaa cgacatggca ttgaaattgg ttggcgggag
     2221 ccaccttata gcgaatataa agacgaccta tcggtctaaa aagcccgcca aaaatttgaa
     2281 gatgcctggt gtatattacg tcgactatag gcttgagaga ataaaagaag ctaacaacga
     2341 aacctatgtg gagcaacatg aggttgcagt tgcacgctac tgcgatctgc cgagcaaatt
     2401 ggggcacaag ctgaattgat cagctagagc tcgctaatca gcctcgactg tgccttctag
     2461 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2521 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2581 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagataatag
     2641 caggcatgct ggggatgcgg tgggctctat ggtctagatt tagggtaggg gaaatgtgcg
     2701 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2761 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2821 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2881 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2941 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     3001 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     3061 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     3121 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     3181 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3241 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3301 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3361 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3421 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3481 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     3541 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3601 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3661 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3721 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3781 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3841 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3901 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3961 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4021 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4081 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4141 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4201 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4261 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4321 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4381 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4441 ctttttacgg ttcctggcct tttgctggcc ttttgctca
//
