LOCUS       sequence-446500-        4928 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 4928)
  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 4928)
  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-gRNA1-EGFP
FEATURES             Location/Qualifiers
     source          1..4928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(72..527)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(209..230)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     421..440
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        554..658
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(659..727,728..1519)
                     /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(877..896)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1690..2278
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2179..2198
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2432..2449
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    2590..2711
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2601..2620)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2655..2674
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(2834..3547,3548..3550,3551..3553))
                     /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(2837..3547,3548..3550,3551..3553))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2879..2900)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3228..3247
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3487..3508
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(3562..4072)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3625..3643)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    4121..4238
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        complement(4290..4365)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4389..4637)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4447..4466)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4617..4637)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    4716..4807
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(4748..4767)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    4821..4869
                     /note="synthetic polyadenylation signal"
ORIGIN
        1 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
       61 tggcgaatgg caaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
      121 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
      181 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
      241 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
      301 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
      361 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
      421 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
      481 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
      541 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      601 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
      661 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
      721 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
      781 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
      841 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      901 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      961 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     1021 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     1081 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     1141 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     1201 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     1261 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     1321 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtcccg
     1381 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     1441 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     1501 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     1561 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     1621 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1681 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1741 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1801 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     1861 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1921 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1981 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     2041 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     2101 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     2161 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2221 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     2281 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2341 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     2401 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     2461 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     2521 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     2581 tcggtcgact aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     2641 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     2701 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
     2761 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatctag
     2821 agtcgcggcc gctttacttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca
     2881 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact
     2941 gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
     3001 gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt
     3061 tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt
     3121 tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct
     3181 cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc
     3241 cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga
     3301 agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca
     3361 gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca
     3421 gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc
     3481 cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc
     3541 ccttgctcac catggatccc cctggggaga gaggtcggtg attcggtcaa cgagggagcc
     3601 gactgccgac gtgcgctccg gaggcttgca gaatgcggaa caccgcgcgg gcaggaacag
     3661 ggcccacact accgccccac accccgcctc ccgcaccgcc ccttcccggc cgctgctctc
     3721 ggcgcgccct gctgagcagc cgctattggc cacagcccat cgcggtcggc gcgctgccat
     3781 tgctccctgg cgctgtccgt ctgcgagggt actagtgaga cgtgcggctt ccgtttgtca
     3841 cgtccggcac gccgcgaacc gcaaggaacc ttcccgactt aggggcggag caggaagcgt
     3901 cgccgggggg cccacaaggg tagcggcgaa gatccgggtg acgctgcgaa cggacgtgaa
     3961 gaatgtgcga gtcccagggt cggcgccgct gcgtttcccg gaaccacgcc cagagcagcc
     4021 gcgtccctgc gcaaacccag ggctgccttg gaaaaggcgc aaccccaacc ccctcgagcc
     4081 gcggccaaag tggatctctg ctgtccctgt aataaacccg aaaattttga atttttgtaa
     4141 tttgtttttg taattcttta gtttgtatgt ctgttgctat tatgtctact attctttccc
     4201 ctgcactgta ccccccaatc cccccttttc ttttaaaatt gtggatgaat actgccattt
     4261 gtctcgaggt cgagaattct ttaaaaaaag caccgactcg gtgccacttt ttcaagttga
     4321 taacggacta gccttatttt aacttgctat ttctagctct aaaaccgccg accaaccaaa
     4381 gctcggccgg tgtttcgtcc tttccacaag atatataaag ccaagaaatc gaaatacttt
     4441 caagttacgg taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac
     4501 ccaagaaatt attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc
     4561 aaattaattc caattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc
     4621 atgggaaata ggccctcggt gaagggggcg gccgctcgag gctagtctcg tgatcgatac
     4681 cgtcgagatc cgttcactaa tcggtaccta tcgatagaga aatgttctgg cacctgcact
     4741 tgcactgggg acagcctatt ttgctagttt gttttgtttc gttttgtttt gatggagagc
     4801 gtatgttagt actatcgatt cacacaaaaa accaacacac agatgtaatg aaaataaaga
     4861 tattttattg cggccgctcc aagtacctcc cgtaccttaa tattacttac ttatcatggt
     4921 agcttggg
//
