LOCUS       sequence-495328-        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   Ataei L, Zhang J, Monis S, Giemza K, Mittal K, Yang J, Shimomura M, 
            McStay B, Wilson MD, Ramalho-Santos M
  TITLE     LINE1 elements at distal junctions of rDNA repeats regulate 
            nucleolar organization in human embryonic stem cells.
  JOURNAL   Genes Dev. 2025 Feb 3;39(3-4):280-298. doi: 10.1101/gad.351979.124.
  PUBMED    39797762
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: "Genes 
            Dev."; date: "2025-02-3"; volume: "39(3-4):280-298. doi"; pages: " 
            10.1101/gad.351979.124"
COMMENT     Sequence Label: rDNA-adj gRNA #1
FEATURES             Location/Qualifiers
     source          1..4927
                     /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(3172..3420)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3230..3249)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3400..3420)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    3499..3590
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(3531..3550)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    3604..3652
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(3783..4238)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3920..3941)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4132..4151
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4265..4369
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4370..4438,4439..4927,1..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(4588..4607)
                     /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 ctctaaaacc ttacagaaag tctcaaatgc cggtgtttcg
     3181 tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat
     3241 atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt
     3301 tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttccaattat
     3361 ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc
     3421 ggtgaagggg gcggccgctc gaggctagtc tcgtgatcga taccgtcgag atccgttcac
     3481 taatcggtac ctatcgatag agaaatgttc tggcacctgc acttgcactg gggacagcct
     3541 attttgctag tttgttttgt ttcgttttgt tttgatggag agcgtatgtt agtactatcg
     3601 attcacacaa aaaaccaaca cacagatgta atgaaaataa agatatttta ttgcggccgc
     3661 tccaagtacc tcccgtacct taatattact tacttatcat ggtagcttgg gctggcgtaa
     3721 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     3781 gcaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3841 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     3901 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     3961 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     4021 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     4081 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     4141 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     4201 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     4261 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4321 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     4381 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     4441 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     4501 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     4561 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     4621 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     4681 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     4741 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     4801 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     4861 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     4921 ggcaaca
//
