LOCUS       sequence-255630-        8878 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PARN gRNA for CRISPRi.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8878)
  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 8878)
  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.PARN-2
FEATURES             Location/Qualifiers
     source          1..8878
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1447..1680
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1865..1909
                     /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    2207..2324
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2577..2610)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2614..2634
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        2880..3091
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          3092..4030
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3987..4007
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4051..4647
                     /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(4051..4070)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4547..4567
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4657..4710
                     /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             4723..5421
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(5441..5474)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5530..6118
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5583..5603)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6001..6012)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6121..6137)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6122..6138)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6647..6827
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6889..7477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6969..6988)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7648..8439,8440..8508))
                     /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     8271..8290
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8509..8613)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8688..8707)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg atcttcagac ctggaggagg
     1321 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     1381 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     1441 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     1501 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     1561 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     1621 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     1681 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     1741 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     1801 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     1861 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     1921 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     1981 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     2041 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
     2101 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
     2161 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     2221 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2281 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2341 acagcagaga tccagtttgg ttagtaccgg gcccgctcta gagatccgac gcgccatctc
     2401 taggcccgcg ccggccccct cgcacggact tgtgggagaa gctcggctac tcccctgccc
     2461 cggttaattt gcatataata tttcctagta actatagagg cttaatgtgc gataaaagac
     2521 agataatctg ttctttttaa tactagctac attttacatg ataggcttgg atttctataa
     2581 cttcgtatag catacattat acgaagttat aaacagcaca aaaggaaact caccctaact
     2641 gtaaagtaat tgtgtgtttt gagactataa gtatcccttg gagaaccacc ttgttggtag
     2701 aaccgctgag gcgggcggtt taagagctaa gctggaaaca gcatagcaag tttaaataag
     2761 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctcgagtact
     2821 aggatccatt aggcggccgc gtggataacc gtattaccgc catgcatgtg cccgtcagtg
     2881 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
     2941 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
     3001 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
     3061 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc
     3121 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta
     3181 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc
     3241 ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg
     3301 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat
     3361 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc
     3421 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg
     3481 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg
     3541 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat
     3601 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg
     3661 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg
     3721 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg
     3781 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcgc gcttttggag
     3841 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg
     3901 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct
     3961 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct
     4021 tccatttcag gtgtcgtgag ctagcccacc atgaccgagt acaagcccac ggtgcgcctc
     4081 gccacccgcg acgacgtccc ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac
     4141 cccgccacgc gccacaccgt cgacccggac cgccacatcg agcgggtcac cgagctgcaa
     4201 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     4261 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     4321 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     4381 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     4441 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     4501 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     4561 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     4621 tggtgcatga cccgcaagcc cggtgccgga tcgggagagg gcagaggaag tctgctaaca
     4681 tgcggtgacg tcgaggagaa tcctggccca ccggtcgcca ccagcgagct gattaaggag
     4741 aacatgcaca tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca
     4801 tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag
     4861 ggcggccctc tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag
     4921 accttcatca accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc
     4981 ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac
     5041 accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca
     5101 tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg
     5161 taccccgctg acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg
     5221 agccatctga tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc
     5281 aagatgcctg gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac
     5341 gagacctacg tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa
     5401 ctggggcaca agcttaatta agaattcgtc gagggaccta ataacttcgt atagcataca
     5461 ttatacgaag ttatacatgt ttaagggttc cggttccact aggtacaatt cgatatcaag
     5521 cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     5581 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     5641 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     5701 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     5761 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     5821 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     5881 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     5941 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     6001 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     6061 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     6121 cgataccgtc gacctcgatc gagacctaga aaaacatgga gcaatcacaa gtagcaatac
     6181 agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt
     6241 tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag
     6301 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
     6361 tatccttgat ctgtggatct accacacaca aggctacttc cctgattggc agaactacac
     6421 accagggcca gggatcagat atccactgac ctttggatgg tgctacaagc tagtaccagt
     6481 tgagcaagag aaggtagaag aagccaatga aggagagaac acccgcttgt tacaccctgt
     6541 gagcctgcat gggatggatg acccggagag agaagtatta gagtggaggt ttgacagccg
     6601 cctagcattt catcacatgg cccgagagct gcatccggac tgtactgggt ctctctggtt
     6661 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     6721 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     6781 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagca tgtgagcaaa
     6841 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     6901 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     6961 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     7021 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     7081 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     7141 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     7201 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     7261 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     7321 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     7381 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     7441 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     7501 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     7561 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     7621 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     7681 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     7741 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     7801 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     7861 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7921 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     7981 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     8041 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     8101 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     8161 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     8221 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     8281 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     8341 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     8401 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     8461 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     8521 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     8581 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     8641 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     8701 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     8761 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     8821 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgtt
//
