LOCUS       sequence-446503-        4927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRi-KD of human Nucleolin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4927)
  AUTHORS   Zhang J, Ataei L, Mittal K, Wu L, Caldwell L, Huynh L, Sarajideen S,
            Tse K, Simon MM, Mazid MA, Cook DP, Trcka D, Kwan T, Hoffman MM, 
            Wrana JL, Esteban MA, Ramalho-Santos M
  TITLE     LINE1 and PRC2 control nucleolar organization and repression of the 
            8C state in human ESCs.
  JOURNAL   Dev Cell. 2024 Oct 14:S1534-5807(24)00574-4. doi: 
            10.1016/j.devcel.2024.09.024.
  PUBMED    39413784
REFERENCE   2  (bases 1 to 4927)
  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"; journalName: "Dev Cell. 
            2024 Oct 14:S1534-5807(24)00574-4. doi: 
            10.1016/j.devcel.2024.09.024."
COMMENT     Sequence Label: pGL3-NCL-gRNA2-EGFP
FEATURES             Location/Qualifiers
     source          1..4927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     323..342
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(561..665)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      692..1147
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(779..798)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     989..1010
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    1278..1326
                     /note="synthetic polyadenylation signal"
     misc_feature    1340..1431
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     1380..1399
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        1510..1758
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1510..1530
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1681..1700
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1781..1856
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    1908..2025
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2074..2584
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2503..2521
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(2593..2595,2596..2598,2599..3312)
                     /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(2593..2595,2596..2598,2599..3309)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2638..2659)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2899..2918)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3246..3267
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3435..3556
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3472..3491)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3526..3545
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3697..3714)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3868..4456)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3948..3967)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4627..4927,1..491,492..560))
                     /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"
ORIGIN
        1 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
       61 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
      121 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
      181 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
      241 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
      301 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
      361 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
      421 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
      481 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
      541 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
      601 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
      661 ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg
      721 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
      781 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
      841 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
      901 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
      961 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1021 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     1081 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     1141 acaatttgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     1201 tcttcgctat tacgccagcc caagctacca tgataagtaa gtaatattaa ggtacgggag
     1261 gtacttggag cggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt
     1321 tgtgtgaatc gatagtacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac
     1381 tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc tatcgatagg
     1441 taccgattag tgaacggatc tcgacggtat cgatcacgag actagcctcg agcggccgcc
     1501 cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     1561 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     1621 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     1681 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     1741 tggaaaggac gaaacaccgg aagaagccaa gcgacggcga gttttagagc tagaaatagc
     1801 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     1861 tttaaagaat tctcgacctc gagacaaatg gcagtattca tccacaattt taaaagaaaa
     1921 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     1981 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     2041 gacagcagag atccactttg gccgcggctc gagggggttg gggttgcgcc ttttccaagg
     2101 cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg
     2161 cgccgaccct gggactcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc
     2221 cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc
     2281 cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct
     2341 cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa
     2401 tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc
     2461 ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa
     2521 gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc
     2581 ccagggggat ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     2641 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     2701 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     2761 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     2821 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     2881 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     2941 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     3001 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     3061 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     3121 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     3181 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     3241 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     3301 ctgtacaagt aaagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg
     3361 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     3421 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     3481 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     3541 tcatcaatgt atcttagtcg accgatgccc ttgagagcct tcaacccagt cagctccttc
     3601 cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa
     3661 ctcgtaggac aggtgccggc agcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3721 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3781 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3841 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3901 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3961 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4021 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4081 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4141 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4201 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4261 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4321 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4381 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4441 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4501 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4561 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4621 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4681 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4741 tggccccagt gctgcaatga taccgcggga cccacgctca ccggctccag atttatcagc
     4801 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4861 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4921 gcgcaac
//
