LOCUS       sequence-495329-        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 #3
FEATURES             Location/Qualifiers
     source          1..4927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      346..934
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     835..854
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1088..1105
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    1246..1367
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1257..1276)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1311..1330
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(1490..2203,2204..2206,2207..2209))
                     /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(1493..2203,2204..2206,2207..2209))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1535..1556)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1884..1903
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2143..2164
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(2218..2728)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2281..2299)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    2777..2894
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        complement(2946..3021)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3044..3292)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3102..3121)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3272..3292)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    3371..3462
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(3403..3422)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    3476..3524
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(3655..4110)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3792..3813)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4004..4023
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4137..4241
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4242..4310,4311..4927,1..175)
                     /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(4460..4479)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tgctgataaa tctggagccg gtgagcgtgg gtcccgcggt atcattgcag cactggggcc
       61 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
      121 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
      181 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
      241 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
      301 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
      361 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
      421 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
      481 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
      541 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
      601 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
      661 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
      721 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
      781 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
      841 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
      901 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
      961 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     1021 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     1081 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc tgccggcacc tgtcctacga
     1141 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     1201 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgactaaga tacattgatg
     1261 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     1321 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     1381 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     1441 acctctacaa atgtggtatg gctgattatg atctagagtc gcggccgctt tacttgtaca
     1501 gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat
     1561 cgcgcttctc gttggggtct ttgctcaggg cggactgggt gctcaggtag tggttgtcgg
     1621 gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca
     1681 cgctgccgtc ctcgatgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct
     1741 tgtcggccat gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga
     1801 tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc
     1861 cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc
     1921 gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt
     1981 cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg
     2041 gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat
     2101 cgccctcgcc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga
     2161 ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg gatccccctg
     2221 gggagagagg tcggtgattc ggtcaacgag ggagccgact gccgacgtgc gctccggagg
     2281 cttgcagaat gcggaacacc gcgcgggcag gaacagggcc cacactaccg ccccacaccc
     2341 cgcctcccgc accgcccctt cccggccgct gctctcggcg cgccctgctg agcagccgct
     2401 attggccaca gcccatcgcg gtcggcgcgc tgccattgct ccctggcgct gtccgtctgc
     2461 gagggtacta gtgagacgtg cggcttccgt ttgtcacgtc cggcacgccg cgaaccgcaa
     2521 ggaaccttcc cgacttaggg gcggagcagg aagcgtcgcc ggggggccca caagggtagc
     2581 ggcgaagatc cgggtgacgc tgcgaacgga cgtgaagaat gtgcgagtcc cagggtcggc
     2641 gccgctgcgt ttcccggaac cacgcccaga gcagccgcgt ccctgcgcaa acccagggct
     2701 gccttggaaa aggcgcaacc ccaaccccct cgagccgcgg ccaaagtgga tctctgctgt
     2761 ccctgtaata aacccgaaaa ttttgaattt ttgtaatttg tttttgtaat tctttagttt
     2821 gtatgtctgt tgctattatg tctactattc tttcccctgc actgtacccc ccaatccccc
     2881 cttttctttt aaaattgtgg atgaatactg ccatttgtct cgaggtcgag aattctttaa
     2941 aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct tattttaact
     3001 tgctatttct agctctaaaa ccactatcag agtcttctgt accggtgttt cgtcctttcc
     3061 acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc atatgatagt
     3121 ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac tttctacgtc
     3181 acgtattttg tactaatatc tttgtgttta cagtcaaatt aattccaatt atctctctaa
     3241 cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc tcggtgaagg
     3301 gggcggccgc tcgaggctag tctcgtgatc gataccgtcg agatccgttc actaatcggt
     3361 acctatcgat agagaaatgt tctggcacct gcacttgcac tggggacagc ctattttgct
     3421 agtttgtttt gtttcgtttt gttttgatgg agagcgtatg ttagtactat cgattcacac
     3481 aaaaaaccaa cacacagatg taatgaaaat aaagatattt tattgcggcc gctccaagta
     3541 cctcccgtac cttaatatta cttacttatc atggtagctt gggctggcgt aatagcgaag
     3601 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcaaattg
     3661 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     3721 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     3781 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     3841 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     3901 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     3961 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     4021 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     4081 ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg
     4141 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4201 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4261 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4321 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4381 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4441 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     4501 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     4561 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     4621 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     4681 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     4741 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     4801 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     4861 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     4921 ggtttat
//
