LOCUS       sequence-446493-        4928 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 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-Ctr2-gRNA-EGFP
FEATURES             Location/Qualifiers
     source          1..4928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      474..1062
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     963..982
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1216..1233
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    1374..1495
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1385..1404)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1439..1458
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(1618..2331,2332..2334,2335..2337))
                     /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(1621..2331,2332..2334,2335..2337))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1663..1684)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2012..2031
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2271..2292
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(2346..2856)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2409..2427)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    2905..3022
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        complement(3074..3149)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3173..3421)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3231..3250)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3401..3421)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    3500..3591
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(3532..3551)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    3605..3653
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(3784..4239)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3921..3942)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4133..4152
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4266..4370
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4371..4439,4440..4928,1..303)
                     /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(4589..4608)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
       61 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
      121 tggtttattg ctgataaatc tggagccggt gagcgtgggt cccgcggtat cattgcagca
      181 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
      241 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
      301 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
      361 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
      421 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
      481 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
      541 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
      601 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
      661 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
      721 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
      781 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
      841 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
      901 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      961 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     1021 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     1081 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     1141 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     1201 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgctg ccggcacctg
     1261 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     1321 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gactaagata
     1381 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     1441 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     1501 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag
     1561 caagtaaaac ctctacaaat gtggtatggc tgattatgat ctagagtcgc ggccgcttta
     1621 cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac
     1681 catgtgatcg cgcttctcgt tggggtcttt gctcagggcg gactgggtgc tcaggtagtg
     1741 gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc
     1801 gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg aagttcacct tgatgccgtt
     1861 cttctgcttg tcggccatga tatagacgtt gtggctgttg tagttgtact ccagcttgtg
     1921 ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag
     1981 ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa
     2041 gatggtgcgc tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt
     2101 catgtggtcg gggtagcggc tgaagcactg cacgccgtag gtcagggtgg tcacgagggt
     2161 gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta
     2221 ggtggcatcg ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc
     2281 cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccatgga
     2341 tccccctggg gagagaggtc ggtgattcgg tcaacgaggg agccgactgc cgacgtgcgc
     2401 tccggaggct tgcagaatgc ggaacaccgc gcgggcagga acagggccca cactaccgcc
     2461 ccacaccccg cctcccgcac cgccccttcc cggccgctgc tctcggcgcg ccctgctgag
     2521 cagccgctat tggccacagc ccatcgcggt cggcgcgctg ccattgctcc ctggcgctgt
     2581 ccgtctgcga gggtactagt gagacgtgcg gcttccgttt gtcacgtccg gcacgccgcg
     2641 aaccgcaagg aaccttcccg acttaggggc ggagcaggaa gcgtcgccgg ggggcccaca
     2701 agggtagcgg cgaagatccg ggtgacgctg cgaacggacg tgaagaatgt gcgagtccca
     2761 gggtcggcgc cgctgcgttt cccggaacca cgcccagagc agccgcgtcc ctgcgcaaac
     2821 ccagggctgc cttggaaaag gcgcaacccc aaccccctcg agccgcggcc aaagtggatc
     2881 tctgctgtcc ctgtaataaa cccgaaaatt ttgaattttt gtaatttgtt tttgtaattc
     2941 tttagtttgt atgtctgttg ctattatgtc tactattctt tcccctgcac tgtacccccc
     3001 aatcccccct tttcttttaa aattgtggat gaatactgcc atttgtctcg aggtcgagaa
     3061 ttctttaaaa aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta
     3121 ttttaacttg ctatttctag ctctaaaacg ctacccaccc ccccatcgca ccggtgtttc
     3181 gtcctttcca caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca
     3241 tatgatagtc cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact
     3301 ttctacgtca cgtattttgt actaatatct ttgtgtttac agtcaaatta attccaatta
     3361 tctctctaac agccttgtat cgtatatgca aatatgaagg aatcatggga aataggccct
     3421 cggtgaaggg ggcggccgct cgaggctagt ctcgtgatcg ataccgtcga gatccgttca
     3481 ctaatcggta cctatcgata gagaaatgtt ctggcacctg cacttgcact ggggacagcc
     3541 tattttgcta gtttgttttg tttcgttttg ttttgatgga gagcgtatgt tagtactatc
     3601 gattcacaca aaaaaccaac acacagatgt aatgaaaata aagatatttt attgcggccg
     3661 ctccaagtac ctcccgtacc ttaatattac ttacttatca tggtagcttg ggctggcgta
     3721 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     3781 ggcaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     3841 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     3901 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     3961 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     4021 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     4081 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     4141 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     4201 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa
     4261 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4321 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4381 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4441 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4501 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4561 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4621 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4681 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4741 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4801 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4861 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     4921 tggcaaca
//
