LOCUS       sequence-446498-        4927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRi-KD of human LINE1.
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-hL1-gRNA2-EGFP
FEATURES             Location/Qualifiers
     source          1..4927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    287..335
                     /note="synthetic polyadenylation signal"
     misc_feature    349..440
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     389..408
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        519..767
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     519..539
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     690..709
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        790..865
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    917..1034
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1083..1593
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1512..1530
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(1602..1604,1605..1607,1608..2321)
                     /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(1602..1604,1605..1607,1608..2318)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1647..1668)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1908..1927)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2255..2276
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2444..2565
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2481..2500)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2535..2554
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2706..2723)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2877..3465)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2957..2976)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3636..4427,4428..4496))
                     /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     4259..4278
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4497..4601)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(4628..4927,1..156)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4715..4734)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(4925..4927,1..19)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
       61 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      121 acgcgaattt taacaaaata ttaacgctta caatttgcca ttcgccattc aggctgcgca
      181 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagccc aagctaccat
      241 gataagtaag taatattaag gtacgggagg tacttggagc ggccgcaata aaatatcttt
      301 attttcatta catctgtgtg ttggtttttt gtgtgaatcg atagtactaa catacgctct
      361 ccatcaaaac aaaacgaaac aaaacaaact agcaaaatag gctgtcccca gtgcaagtgc
      421 aggtgccaga acatttctct atcgataggt accgattagt gaacggatct cgacggtatc
      481 gatcacgaga ctagcctcga gcggccgccc ccttcaccga gggcctattt cccatgattc
      541 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
      601 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
      661 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
      721 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggc ttaggtaaac
      781 aaagcagcag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
      841 tgaaaaagtg gcaccgagtc ggtgcttttt ttaaagaatt ctcgacctcg agacaaatgg
      901 cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa
      961 gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa
     1021 aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg ccgcggctcg
     1081 agggggttgg ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc
     1141 tctgggcgtg gttccgggaa acgcagcggc gccgaccctg ggactcgcac attcttcacg
     1201 tccgttcgca gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt
     1261 cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa
     1321 acggaagccg cacgtctcac tagtaccctc gcagacggac agcgccaggg agcaatggca
     1381 gcgcgccgac cgcgatgggc tgtggccaat agcggctgct cagcagggcg cgccgagagc
     1441 agcggccggg aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc
     1501 ctgcccgcgc ggtgttccgc attctgcaag cctccggagc gcacgtcggc agtcggctcc
     1561 ctcgttgacc gaatcaccga cctctctccc cagggggatc catggtgagc aagggcgagg
     1621 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1681 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     1741 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     1801 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1861 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     1921 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1981 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     2041 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     2101 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     2161 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     2221 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     2281 ccgccgggat cactctcggc atggacgagc tgtacaagta aagcggccgc gactctagat
     2341 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     2401 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     2461 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     2521 actgcattct agttgtggtt tgtccaaact catcaatgta tcttagtcga ccgatgccct
     2581 tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg
     2641 cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc
     2701 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2761 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2821 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2881 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2941 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3001 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3061 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3121 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3181 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3241 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3301 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3361 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3421 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3481 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3541 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     3601 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3661 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3721 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgggac
     3781 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3841 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3901 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3961 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4021 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4081 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4141 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4201 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4261 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4321 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4381 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4441 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4501 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4561 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4621 ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4681 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4741 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4801 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     4861 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4921 aatagtg
//
