LOCUS       sequence-254142-        8877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CYRANO gRNA for CRISPRi.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8877)
  AUTHORS   Kleaveland B, Shi CY, Stefano J, Bartel DP
  TITLE     A Network of Noncoding Regulatory RNAs Acts in the Mammalian Brain.
  JOURNAL   Cell. 2018 Jul 12;174(2):350-362.e17. doi: 
            10.1016/j.cell.2018.05.022. Epub 2018 Jun 7.
  PUBMED    29887379
REFERENCE   2  (bases 1 to 8877)
  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: 
            "10.1016/j.cell.2018.05.022"; journalName: "Cell"; date: 
            "2018-07-12- 12"; volume: "174"; issue: "2"; pages: "350-362.e17"
COMMENT     Sequence Label: pU6-sgRNA EF1Alpha-puro-T2A-BFP.CYRANO
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        225..423
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     378..398
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             441..621
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    668..793
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1290..1523
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1708..1752
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2050..2167
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2420..2453)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2457..2477
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        2722..2933
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          2934..3872
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3829..3849
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3893..4489
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3893..3912)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4389..4409
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4499..4552
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4565..5263
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(5283..5316)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5372..5960
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5425..5445)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5843..5854)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5963..5979)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5964..5980)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6489..6669
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6731..7319)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6811..6830)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7490..8281,8282..8350))
                     /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     8113..8132
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8351..8455)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8530..8549)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8721..8877,1..223)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
       61 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      121 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      181 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
      241 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
      301 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
      361 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      421 cagcgcgttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct
      481 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
      541 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
      601 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
      661 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
      721 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
      781 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
      841 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
      901 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
      961 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     1021 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     1081 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg
     1141 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     1201 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     1261 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     1321 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     1381 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     1441 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     1501 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     1561 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     1621 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     1681 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     1741 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     1801 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     1861 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     1921 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     1981 attcgattag tgaacggatc ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt
     2041 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     2101 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     2161 aaattttcgg gtttattaca gggacagcag agatccagtt tggttagtac cgggcccgct
     2221 ctagagatcc gacgcgccat ctctaggccc gcgccggccc cctcgcacgg acttgtggga
     2281 gaagctcggc tactcccctg ccccggttaa tttgcatata atatttccta gtaactatag
     2341 aggcttaatg tgcgataaaa gacagataat ctgttctttt taatactagc tacattttac
     2401 atgataggct tggatttcta taacttcgta tagcatacat tatacgaagt tataaacagc
     2461 acaaaaggaa actcacccta actgtaaagt aattgtgtgt tttgagacta taagtatccc
     2521 ttggagaacc accttgttgg cccagaggcc agtttgcagg tttaagagct aagctggaaa
     2581 cagcatagca agtttaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     2641 ggtgcttttt ttctcgagta ctaggatcca ttaggcggcc gcgtggataa ccgtattacc
     2701 gccatgcatg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
     2761 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
     2821 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
     2881 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg
     2941 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa
     3001 ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt
     3061 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct
     3121 ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg
     3181 ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct
     3241 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg
     3301 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc
     3361 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc
     3421 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac
     3481 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga
     3541 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc
     3601 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg
     3661 attagttctc gcgcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga
     3721 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt
     3781 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga
     3841 cagtggttca aagttttttt cttccatttc aggtgtcgtg agctagccca ccatgaccga
     3901 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct
     3961 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat
     4021 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     4081 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     4141 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     4201 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     4261 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     4321 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     4381 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     4441 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gatcgggaga
     4501 gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc caccggtcgc
     4561 caccagcgag ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga
     4621 caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac
     4681 catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac
     4741 tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt
     4801 caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg
     4861 cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa
     4921 gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg
     4981 ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat
     5041 ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc
     5101 caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga
     5161 aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag
     5221 atactgcgac ctccctagca aactggggca caagcttaat taagaattcg tcgagggacc
     5281 taataacttc gtatagcata cattatacga agttatacat gtttaagggt tccggttcca
     5341 ctaggtacaa ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga
     5401 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     5461 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     5521 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     5581 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     5641 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     5701 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     5761 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     5821 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     5881 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     5941 cctttgggcc gcctccccgc atcgataccg tcgacctcga tcgagaccta gaaaaacatg
     6001 gagcaatcac aagtagcaat acagcagcta ccaatgctga ttgtgcctgg ctagaagcac
     6061 aagaggagga ggaggtgggt tttccagtca cacctcaggt acctttaaga ccaatgactt
     6121 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     6181 ttcactccca acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact
     6241 tccctgattg gcagaactac acaccagggc cagggatcag atatccactg acctttggat
     6301 ggtgctacaa gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga
     6361 acacccgctt gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat
     6421 tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg
     6481 actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     6541 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     6601 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     6661 tctctagcag catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6721 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6781 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6841 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6901 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6961 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     7021 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     7081 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     7141 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     7201 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     7261 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     7321 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     7381 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     7441 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     7501 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     7561 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     7621 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     7681 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7741 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7801 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7861 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7921 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7981 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     8041 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     8101 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     8161 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     8221 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     8281 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     8341 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     8401 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     8461 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
     8521 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     8581 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     8641 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
     8701 acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac
     8761 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
     8821 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgt
//
