LOCUS       sequence-173715-        8122 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Designed for genomic deletion screening of functional lncRNAs (long 
            non-coding RNAs) using lentiviral pgRNAs (paired-guide RNAs)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8122)
  AUTHORS   Zhu S, Li W, Liu J, Chen CH, Liao Q, Xu P, Xu H, Xiao T, Cao Z, Peng
            J, Yuan P, Brown M, Liu XS, Wei W
  TITLE     Genome-scale deletion screening of human long non-coding RNAs using 
            a paired-guide RNA CRISPR-Cas9 library.
  JOURNAL   Nat Biotechnol. 2016 Dec;34(12):1279-1286. doi: 10.1038/nbt.3715. 
            Epub 2016 Oct 31.
  PUBMED    27798563
REFERENCE   2  (bases 1 to 8122)
  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: "10.1038/nbt.3715"; 
            journalName: "Nat Biotechnol"; date: "2016-12"; volume: "34"; issue:
            "12"; pages: "1279-1286"
COMMENT     Sequence Label: Wei lab paired-guide RNA (pgRNA) CRISPR–Cas9 l...
FEATURES             Location/Qualifiers
     source          1..8122
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1686..1919
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2104..2148
                     /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    2446..2563
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2643..2890
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     gap             2892..2911
                     /estimated_length=20
     promoter        3059..3307
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3059..3079
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     gap             3309..3328
                     /estimated_length=20
     primer_bind     3425..3441
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3483..3516)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        3632..3935
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3936..4139
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      4178..4187
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4184..4186,4187..4189,4190..4903)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4184..4186,4187..4189,4190..4900)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4229..4250)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4490..4509)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4837..4858
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4922..4955)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5011..5599
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5064..5084)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5482..5493)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5602..5618)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             6128..6308
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6370..6958)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6450..6469)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7129..7920,7921..7989))
                     /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     7752..7771
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7990..8094)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgcgctga tcttcagacc tggaggagga
     1561 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     1621 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     1681 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     1741 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     1801 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1861 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1921 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1981 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     2041 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     2101 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     2161 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     2221 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     2281 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     2341 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
     2401 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
     2461 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2521 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2581 cagcagagat ccagtttggt tagtaccggg cccgctctag aggtaccaag gtcgggcagg
     2641 aagagggcct atttccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     2701 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     2761 aaagtaataa tttcttgggt atttgcagtt tttaaaatta tgttttaaaa tggactatca
     2821 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     2881 acgaaacacc gnnnnnnnnn nnnnnnnnnn ngtttaagag ctatgctgga aacagcatag
     2941 caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     3001 tttttcccgt atctcacgct cttcatcggc gtctagaggt accaaggtct ggcaggaaga
     3061 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     3121 aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
     3181 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     3241 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
     3301 aaacaccgnn nnnnnnnnnn nnnnnnnngt ttaagagcta tgctggaaac agcatagcaa
     3361 gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     3421 ttctcgaggt cgacggtatc gataagctcg cttcacgaga ttccagcagg tcgagggacc
     3481 taataacttc gtatagcata cattatacga agttatatta agggttccaa gcttaagcgg
     3541 ccgcgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt
     3601 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     3661 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     3721 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     3781 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     3841 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     3901 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     3961 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     4021 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     4081 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
     4141 gtttagtgaa ccgtcagatc cgctagcgct accggtcgcc accatggtga gcaagggcga
     4201 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     4261 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     4321 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     4381 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     4441 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     4501 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     4561 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     4621 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     4681 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     4741 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     4801 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     4861 cgccgccggg atcactctcg gcatggacga gctgtacaag tagaattcgt cgagggacct
     4921 aataacttcg tatagcatac attatacgaa gttatacatg tttaagggtt ccggttccac
     4981 taggtacaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     5041 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     5101 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     5161 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     5221 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     5281 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     5341 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     5401 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     5461 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     5521 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     5581 ctttgggccg cctccccgca tcgataccgt cgacctcgat cgagacctag aaaaacatgg
     5641 agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc tagaagcaca
     5701 agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac caatgactta
     5761 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     5821 tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac aaggctactt
     5881 ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga cctttggatg
     5941 gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa
     6001 cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtatt
     6061 agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc tgcatccgga
     6121 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     6181 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     6241 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     6301 ctctagcagc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     6361 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     6421 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6481 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     6541 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6601 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6661 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6721 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6781 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6841 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6901 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6961 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     7021 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     7081 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     7141 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     7201 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     7261 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     7321 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     7381 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     7441 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     7501 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     7561 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     7621 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     7681 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     7741 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     7801 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     7861 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7921 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7981 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     8041 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     8101 tccccgaaaa gtgccacctg ac
//
