LOCUS       sequence-446491-        4927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative control for CRISPRi-KD.
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-Ctr1-gRNA-EGFP
FEATURES             Location/Qualifiers
     source          1..4927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(113..132)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      926..1514
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1415..1434
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1668..1685
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    1826..1947
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1837..1856)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1891..1910
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(2070..2783,2784..2786,2787..2789))
                     /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(2073..2783,2784..2786,2787..2789))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2115..2136)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2464..2483
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2723..2744
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(2798..3308)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2861..2879)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    3357..3474
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        complement(3526..3601)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3624..3872)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3682..3701)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3852..3872)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    3951..4042
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(3983..4002)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    4056..4104
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(4235..4690)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4372..4393)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4584..4603
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4717..4821
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4822..4890,4891..4927,1..755)
                     /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 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
       61 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
      121 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
      181 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
      241 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
      301 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
      361 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
      421 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
      481 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
      541 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
      601 gtgagcgtgg gtcccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
      661 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
      721 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
      781 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
      841 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
      901 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      961 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     1021 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     1081 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     1141 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     1201 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     1261 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     1321 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     1381 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     1441 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     1501 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     1561 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     1621 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     1681 gcgaggaagc ggaagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     1741 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     1801 aaggctctca agggcatcgg tcgactaaga tacattgatg agtttggaca aaccacaact
     1861 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     1921 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     1981 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg
     2041 gctgattatg atctagagtc gcggccgctt tacttgtaca gctcgtccat gccgagagtg
     2101 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct
     2161 ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg
     2221 ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg
     2281 tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg
     2341 ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag
     2401 tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg
     2461 cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg
     2521 ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac
     2581 tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg
     2641 gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac
     2701 acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg
     2761 gtgaacagct cctcgccctt gctcaccatg gatccccctg gggagagagg tcggtgattc
     2821 ggtcaacgag ggagccgact gccgacgtgc gctccggagg cttgcagaat gcggaacacc
     2881 gcgcgggcag gaacagggcc cacactaccg ccccacaccc cgcctcccgc accgcccctt
     2941 cccggccgct gctctcggcg cgccctgctg agcagccgct attggccaca gcccatcgcg
     3001 gtcggcgcgc tgccattgct ccctggcgct gtccgtctgc gagggtacta gtgagacgtg
     3061 cggcttccgt ttgtcacgtc cggcacgccg cgaaccgcaa ggaaccttcc cgacttaggg
     3121 gcggagcagg aagcgtcgcc ggggggccca caagggtagc ggcgaagatc cgggtgacgc
     3181 tgcgaacgga cgtgaagaat gtgcgagtcc cagggtcggc gccgctgcgt ttcccggaac
     3241 cacgcccaga gcagccgcgt ccctgcgcaa acccagggct gccttggaaa aggcgcaacc
     3301 ccaaccccct cgagccgcgg ccaaagtgga tctctgctgt ccctgtaata aacccgaaaa
     3361 ttttgaattt ttgtaatttg tttttgtaat tctttagttt gtatgtctgt tgctattatg
     3421 tctactattc tttcccctgc actgtacccc ccaatccccc cttttctttt aaaattgtgg
     3481 atgaatactg ccatttgtct cgaggtcgag aattctttaa aaaaagcacc gactcggtgc
     3541 cactttttca agttgataac ggactagcct tattttaact tgctatttct agctctaaaa
     3601 cccgtcaggg cacgtttggc gccggtgttt cgtcctttcc acaagatata taaagccaag
     3661 aaatcgaaat actttcaagt tacggtaagc atatgatagt ccattttaaa acataatttt
     3721 aaaactgcaa actacccaag aaattattac tttctacgtc acgtattttg tactaatatc
     3781 tttgtgttta cagtcaaatt aattccaatt atctctctaa cagccttgta tcgtatatgc
     3841 aaatatgaag gaatcatggg aaataggccc tcggtgaagg gggcggccgc tcgaggctag
     3901 tctcgtgatc gataccgtcg agatccgttc actaatcggt acctatcgat agagaaatgt
     3961 tctggcacct gcacttgcac tggggacagc ctattttgct agtttgtttt gtttcgtttt
     4021 gttttgatgg agagcgtatg ttagtactat cgattcacac aaaaaaccaa cacacagatg
     4081 taatgaaaat aaagatattt tattgcggcc gctccaagta cctcccgtac cttaatatta
     4141 cttacttatc atggtagctt gggctggcgt aatagcgaag aggcccgcac cgatcgccct
     4201 tcccaacagt tgcgcagcct gaatggcgaa tggcaaattg taagcgttaa tattttgtta
     4261 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     4321 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     4381 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     4441 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     4501 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     4561 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     4621 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     4681 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4741 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4801 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     4861 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     4921 gctgaag
//
