LOCUS       sequence-181079-        7721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  construction of paired sgRNAs driven by two U6 promoters. Vector 
            itself only has one U6 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7721)
  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 7721)
  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: scaffold-linker-U6
FEATURES             Location/Qualifiers
     source          1..7721
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     185..204
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     444..465
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      507..516
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(555..758)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        759..1062
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    1178..1211
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(1275..1523)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1333..1352)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1503..1523)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    1731..1848
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(2146..2190)
                     /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    2375..2608
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    3105..3230
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             3277..3457
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(3475..3673)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        3675..4054
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4226..4245
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4320..4424
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4425..4493,4494..5285)
                     /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(4643..4662)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5456..6044
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5945..5964
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     LTR             6106..6286
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     6796..6812
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    6815..7403
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             6921..6932
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7330..7350
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    7459..7492
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(7512..7721,1..504,505..507,508..510))
                     /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             complement(join(7515..7721,1..504,505..507,508..510))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7557..7578)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac
       61 gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa
      121 gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc
      181 gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc
      241 gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca
      301 ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt
      361 ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga
      421 cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc
      481 ggtgaacagc tcctcgccct tgctcaccat ggtggcgacc ggtagcgcta gcggatctga
      541 cggttcacta aaccagctct gcttatatag acctcccacc gtacacgcct accgcccatt
      601 tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa
      661 aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc
      721 tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat
      781 acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag
      841 gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga
      901 tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc
      961 cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg
     1021 ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg
     1081 ctatgaacta atgaccccgt aattgattac tattaataac taatgcatgg cggtaatacg
     1141 gttatccacg cggccgctta agcttggaac ccttaatata acttcgtata atgtatgcta
     1201 tacgaagtta ttaggtccct cgacctgctg gaatctcgtg aagcgagctt atcgataccg
     1261 tcgacccgtc tcacggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa
     1321 tactttcaag ttacggtaag catatgatag tccattttaa aacataattt taaaactgca
     1381 aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt
     1441 acagtcaaat taattctaat tatctctcta acagccttgt atcgtatatg caaatatgaa
     1501 ggaatcatgg gaaataggcc ctcttcctgc cagaccttgg tacctctaga cgccgatgaa
     1561 gagcgtgaga tacgggaaaa aaaagcaccg actcggtgcc actttttcaa gttgataacg
     1621 gactagcctt atttaaactt gctatgctgt ttccagcata gctcttaaac tgagacgagc
     1681 gggcccggta ctaaccaaac tggatctctg ctgtccctgt aataaacccg aaaattttga
     1741 atttttgtaa tttgtttttg taattcttta gtttgtatgt ctgttgctat tatgtctact
     1801 attctttccc ctgcactgta ccccccaatc cccccttttc ttttaaaatt gtggatgaat
     1861 actgccattt gtctgcagaa ttggcgcacg cagtgccgat ccgttcacta atcgaatgga
     1921 tctgtctctg tctctctctc caccttcttc ttctattcct tcgggcctgt cgggtcccct
     1981 cggggttggg aggtgggtct gaaacgataa tggtgaatat ccctgcctaa ctctattcac
     2041 tatagaaagt acagcaaaaa ctattcttaa acctaccaag cctcctacta tcattatgaa
     2101 taattttata taccacagcc aatttgttat gttaaaccaa ttccacaaac ttgcccattt
     2161 atctaattcc aataattctt gttcattctt ttcttgctgg ttttgcgatt cttcaattaa
     2221 ggagtgtatt aagcttgtgt aattgttaat ttctctgtcc cactccatcc aggtcgtgtg
     2281 attccaaatc tgttccagag atttattact ccaactagca ttccaaggca cagcagtggt
     2341 gcaaatgagt tttccagagc aaccccaaat ccccaggagc tgttgatcct ttaggtatct
     2401 ttccacagcc aggattcttg cctggagctg cttgatgccc cagactgtga gttgcaacag
     2461 atgctgttgc gcctcaatag ccctcagcaa attgttctgc tgctgcacta taccagacaa
     2521 taattgtctg gcctgtaccg tcagcgtcat tgacgctgcg cccatagtgc ttcctgctgc
     2581 tcccaagaac ccaaggaaca aagctcctat tcccactgct cttttttctc tctgcaccac
     2641 tcttctcttt gccttggtgg gtgctactcc taatggttca atttttacta ctttatattt
     2701 atataattca cttctccaat tgtccctcat atctcctcct ccaggtctga agatcagcgg
     2761 ccggccgctt gctgtgcggt ggtcttactt ttgttttgct cttcctctat cttgtctaaa
     2821 gcttccttgg tgtcttttat ctctatcctt tgatgcacac aatagagggt tgctactgta
     2881 ttatataatg atctaagttc ttctgatcct gtctgaaggg atggttgtag ctgtcccagt
     2941 atttgtctac agccttctga tgtttctaac aggccaggat taactgcgaa tcgttctagc
     3001 tccctgcttg cccatactat atgttttaat ttatattttt tctttccccc tggccttaac
     3061 cgaatttttt cccatcgcga tctaattctc ccccgcttaa tactgacgct ctcgcaccca
     3121 tctctctcct tctagcctcc gctagtcaaa atttttggcg tactcaccag tcgccgcccc
     3181 tcgcctcttg ccgtgcgcgc ttcagcaagc cgagtcctgc gtcgagagag ctcctctggt
     3241 ttccctttcg ctttcaagtc cctgttcggg cgccactgct agagattttc cacactgact
     3301 aaaagggtct gagggatctc tagttaccag agtcacacaa cagacgggca cacactactt
     3361 gaagcactca aggcaagctt tattgaggct taagcagtgg gttccctagt tagccagaga
     3421 gctcccaggc tcagatctgg tctaaccaga gagacccagt acaggcaaaa cgcgctgctt
     3481 atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc ggagttgtta
     3541 cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg
     3601 gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt gatgtactgc
     3661 caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga
     3721 aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg
     3781 tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
     3841 cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca
     3901 tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt
     3961 accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt
     4021 gattactatt aataactagt caataatcaa tgtcaacgcg tatatctggc ccgtacatcg
     4081 cgaagcagcg caaaacgcct aaccctaagc agattcttca tgcaattgtc ggtcaagcct
     4141 tgccttgttg tagcttaaat tttgctcgcg cactactcag cgacctccaa cacacaagca
     4201 gggagcagat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     4261 aggggatcgg gagatctccc gatccgtcga cgtcaggtgg cacttttcgg ggaaatgtgc
     4321 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4381 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4441 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4501 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4561 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4621 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     4681 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     4741 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4801 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     4861 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4921 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     4981 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5041 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5101 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5161 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5221 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5281 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5341 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5401 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5461 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5521 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5581 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     5641 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5701 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5761 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5821 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5881 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5941 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6001 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6061 cctttttacg gttcctggcc ttttgctggc cttttgctca catgctgcta gagattttcc
     6121 acactgacta aaagggtctg agggatctct agttaccaga gtcacacaac agacgggcac
     6181 acactacttg aagcactcaa ggcaagcttt attgaggctt aagcagtggg ttccctagtt
     6241 agccagagag ctcccaggct cagatctggt ctaaccagag agacccagta cagtccggat
     6301 gcagctctcg ggccatgtga tgaaatgcta ggcggctgtc aaacctccac tctaatactt
     6361 ctctctccgg gtcatccatc ccatgcaggc tcacagggtg taacaagcgg gtgttctctc
     6421 cttcattggc ttcttctacc ttctcttgct caactggtac tagcttgtag caccatccaa
     6481 aggtcagtgg atatctgatc cctggccctg gtgtgtagtt ctgccaatca gggaagtagc
     6541 cttgtgtgtg gtagatccac agatcaagga tatcttgtct tcgttgggag tgaattagcc
     6601 cttccagtcc ccccttttct tttaaaaagt ggctaagatc tacagctgcc ttgtaagtca
     6661 ttggtcttaa aggtacctga ggtgtgactg gaaaacccac ctcctcctcc tcttgtgctt
     6721 ctagccaggc acaatcagca ttggtagctg ctgtattgct acttgtgatt gctccatgtt
     6781 tttctaggtc tcgatcgagg tcgacggtat cgatgcgggg aggcggccca aagggagatc
     6841 cgactcgtct gagggcgaag gcgaagacgc ggaagaggcc gcagagccgg cagcaggccg
     6901 cgggaaggaa ggtccgctgg attgagggcc gaagggacgt agcagaagga cgtcccgcgc
     6961 agaatccagg tggcaacaca ggcgagcagc caaggaaagg acgatgattt ccccgacaac
     7021 accacggaat tgtcagtgcc caacagccga gcccctgtcc agcagcgggc aaggcaggcg
     7081 gcgatgagtt ccgccgtggc aatagggagg gggaaagcga aagtcccgga aaggagctga
     7141 caggtggtgg caatgcccca accagtgggg gttgcgtcag caaacacagt gcacaccacg
     7201 ccacgttgcc tgacaacggg ccacaactcc tcataaagag acagcaacca ggatttatac
     7261 aaggaggaga aaatgaaagc catacgggaa gcaatagcat gatacaaagg cattaaagca
     7321 gcgtatccac atagcgtaaa aggagcaaca tagttaagaa taccagtcaa tctttcacaa
     7381 attttgtaat ccagaggttg attatcgata agcttgatat cgaattgtac ctagtggaac
     7441 cggaaccctt aaacatgtat aacttcgtat aatgtatgct atacgaagtt attaggtccc
     7501 tcgacgaatt cctacttgta cagctcgtcc atgccgagag tgatcccggc ggcggtcacg
     7561 aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag ggcggactgg
     7621 gtgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg ggtgttctgc
     7681 tggtagtggt cggcgagctg cacgctgccg tcctcgatgt t
//
