LOCUS       sequence-445989-        4384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP targeting crRNA for RfxCas13d expressed from hU6-2xTetO 
            promoter and non-target BFP protein expressed from EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4384)
  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 4384)
  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-crEGFP_EF1a-BFP
FEATURES             Location/Qualifiers
     source          1..4384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        46..81
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        105..140
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        195..1373
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          426..1364
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1321..1341
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(2116..2133)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2122..2346
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2373..2477
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2478..2546,2547..3338)
                     /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(2696..2715)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3509..4097
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3998..4017
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4160..4180
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4331..4350
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    4361..4379
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 tatctcccta tcagtgatag acaccgtgga aaggacgaaa caccgcaagt aaacccctac
       61 caactggtcg gggtttgaaa ccatgatata gacgttgtgg ctgtcaagta aacccctacc
      121 aactggtcgg ggtttgaaac tttttttttc acctatgtat cgtatcaagt actagaactc
      181 cccagtggga attcggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
      241 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
      301 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
      361 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
      421 cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt
      481 gcgtgccttg aattacttcc acctggctgc agtacgtgat tcttgatccc gagcttcggg
      541 ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt
      601 gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg
      661 cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga
      721 cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt
      781 cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga
      841 ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc
      901 ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg
      961 cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga
     1021 gctcaaaatg aaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga
     1081 aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt
     1141 ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg
     1201 ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt
     1261 ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc
     1321 tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgacgtacgg
     1381 ccaccatgag catgtccgaa ctgattaaag agaacatgca catgaaactt tatatggaag
     1441 gtacggtcga caaccaccat ttcaaatgta cctccgaagg agagggcaaa ccctacgagg
     1501 gcacccaaac gatgagaatt aaagtagtag aaggtgggcc gctgcccttt gcctttgaca
     1561 ttctggcaac ttcttttctc tacgggagta aaacttttat caaccatacg caaggcattc
     1621 cagacttctt taagcagtca tttcccgagg ggtttacttg ggagcgggtc acaacatacg
     1681 aggacggcgg cgtgctgacc gctactcagg acaccagcct gcaagacggg tgtttgatct
     1741 acaatgtgaa gattcgaggc gtaaacttta cttcaaacgg cccagttatg cagaaaaaaa
     1801 ccctcggctg ggaggctttt acagagactc tttacccagc cgacggtggt ttggaaggcc
     1861 gaaacgacat ggcattgaaa ttggttggcg ggagccacct tatagcgaat ataaagacga
     1921 cctatcggtc taaaaagccc gccaaaaatt tgaagatgcc tggtgtatat tacgtcgact
     1981 ataggcttga gagaataaaa gaagctaaca acgaaaccta tgtggagcaa catgaggttg
     2041 cagttgcacg ctactgcgat ctgccgagca aattggggca caagctgaat tgatcagcta
     2101 gagctcgcta atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2161 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2221 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2281 aggacagcaa gggggaggat tgggaagata atagcaggca tgctggggat gcggtgggct
     2341 ctatggtcta gatttagggt aggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2401 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2461 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     2521 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     2581 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     2641 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     2701 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     2761 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     2821 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     2881 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     2941 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3001 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3061 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3121 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3181 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3241 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3301 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3361 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3421 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3481 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3541 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3601 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     3661 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3721 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3781 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3841 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3901 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3961 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4021 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4081 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4141 ggccttttgc tcagctagcg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     4201 atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa agatattagt
     4261 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     4321 ttttaaaatg gactatcata tgcttaccgt aacttgaaac tccctatcag tgatagagat
     4381 tata
//
