LOCUS       sequence-445990-        4364 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BFP targeting crRNA for RfxCas13d expressed from hU6 promoter and 
            target BFP protein expressed from EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4364)
  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 4364)
  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-crBFP_EF1a-BFP
FEATURES             Location/Qualifiers
     source          1..4364
                     /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        316..351
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        406..1584
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          637..1575
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1532..1552
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(2327..2344)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2333..2557
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2584..2688
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2689..2757,2758..3549)
                     /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(2907..2926)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3720..4308
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4209..4228
                     /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 acttgaagta
      301 aagtttacgc ctcgacaagt aaacccctac caactggtcg gggtttgaaa cttttttttt
      361 cacctatgta tcgtatcaag tactagaact ccccagtggg aattcggctc cggtgcccgt
      421 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
      481 tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
      541 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
      601 gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc
      661 gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc cacctggctg
      721 cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct
      781 tgcgcttaag gagccccttc gcctcgtgct tgagttgagg cctggcctgg gcgctggggc
      841 cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga taagtctcta
      901 gccatttaaa atttttgatg acctgctgcg acgctttttt tctggcaaga tagtcttgta
      961 aatgcgggcc aagatctgca cactggtatt tcggtttttg gggccgcggg cggcgacggg
     1021 gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga
     1081 atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg
     1141 tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa
     1201 agatggccgc ttcccggccc tgctgcaggg agctcaaaat gaaggacgcg gcgctcggga
     1261 gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct
     1321 tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt
     1381 tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt tccccacact
     1441 gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc cttggaattt
     1501 gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt tcaaagtttt
     1561 tttcttccat ttcaggtgtc gtgacgtacg gccaccatga gcatgtccga actgattaaa
     1621 gagaacatgc acatgaaact ttatatggaa ggtacggtcg acaaccacca tttcaaatgt
     1681 acctccgaag gagagggcaa accctacgag ggcacccaaa cgatgagaat taaagtagta
     1741 gaaggtgggc cgctgccctt tgcctttgac attctggcaa cttcttttct ctacgggagt
     1801 aaaactttta tcaaccatac gcaaggcatt ccagacttct ttaagcagtc atttcccgag
     1861 gggtttactt gggagcgggt cacaacatac gaggacggcg gcgtgctgac cgctactcag
     1921 gacaccagcc tgcaagacgg gtgtttgatc tacaatgtga agattcgagg cgtaaacttt
     1981 acttcaaacg gcccagttat gcagaaaaaa accctcggct gggaggcttt tacagagact
     2041 ctttacccag ccgacggtgg tttggaaggc cgaaacgaca tggcattgaa attggttggc
     2101 gggagccacc ttatagcgaa tataaagacg acctatcggt ctaaaaagcc cgccaaaaat
     2161 ttgaagatgc ctggtgtata ttacgtcgac tataggcttg agagaataaa agaagctaac
     2221 aacgaaacct atgtggagca acatgaggtt gcagttgcac gctactgcga tctgccgagc
     2281 aaattggggc acaagctgaa ttgatcagct agagctcgct aatcagcctc gactgtgcct
     2341 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     2401 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     2461 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagat
     2521 aatagcaggc atgctgggga tgcggtgggc tctatggtct agatttaggg taggggaaat
     2581 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2641 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2701 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2761 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2821 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     2881 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     2941 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     3001 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     3061 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     3121 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     3181 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     3241 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     3301 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     3361 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt
     3421 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     3481 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     3541 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3601 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3661 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3721 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3781 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3841 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     3901 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3961 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4021 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4081 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4141 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4201 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4261 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4321 gcggcctttt tacggttcct ggccttttgc tggccttttg ctca
//
