LOCUS       sequence-495327-        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: IR InterIR gRNA #5
FEATURES             Location/Qualifiers
     source          1..4927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     581..600
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(819..923)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      950..1405
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1037..1056)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1247..1268
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    1536..1584
                     /note="synthetic polyadenylation signal"
     misc_feature    1598..1689
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     1638..1657
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        1768..2016
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1768..1788
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1939..1958
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2039..2114
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2166..2283
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2332..2842
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2761..2779
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(2851..2853,2854..2856,2857..3570)
                     /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(2851..2853,2854..2856,2857..3567)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2896..2917)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3157..3176)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3504..3525
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3693..3814
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3730..3749)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3784..3803
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3955..3972)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4126..4714)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4206..4225)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4885..4927,1..749,750..818))
                     /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 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
       61 ggcttaccat ctggccccag tgctgcaatg ataccgcggg acccacgctc accggctcca
      121 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
      181 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
      241 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
      301 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
      361 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
      421 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
      481 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
      541 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
      601 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
      661 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
      721 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
      781 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
      841 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
      901 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt
      961 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     1021 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     1081 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     1141 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     1201 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     1261 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     1321 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     1381 aacaaaatat taacgcttac aatttgccat tcgccattca ggctgcgcaa ctgttgggaa
     1441 gggcgatcgg tgcgggcctc ttcgctatta cgccagccca agctaccatg ataagtaagt
     1501 aatattaagg tacgggaggt acttggagcg gccgcaataa aatatcttta ttttcattac
     1561 atctgtgtgt tggttttttg tgtgaatcga tagtactaac atacgctctc catcaaaaca
     1621 aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca ggtgccagaa
     1681 catttctcta tcgataggta ccgattagtg aacggatctc gacggtatcg atcacgagac
     1741 tagcctcgag cggccgcccc cttcaccgag ggcctatttc ccatgattcc ttcatatttg
     1801 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
     1861 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     1921 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     1981 ttggctttat atatcttgtg gaaaggacga aacaccggag tctgggttac agaacacagt
     2041 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     2101 caccgagtcg gtgctttttt taaagaattc tcgacctcga gacaaatggc agtattcatc
     2161 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     2221 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     2281 tttcgggttt attacaggga cagcagagat ccactttggc cgcggctcga gggggttggg
     2341 gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg
     2401 ttccgggaaa cgcagcggcg ccgaccctgg gactcgcaca ttcttcacgt ccgttcgcag
     2461 cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc
     2521 cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc
     2581 acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc
     2641 gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga
     2701 aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg
     2761 gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg
     2821 aatcaccgac ctctctcccc agggggatcc atggtgagca agggcgagga gctgttcacc
     2881 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2941 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     3001 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     3061 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     3121 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     3181 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     3241 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     3301 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     3361 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     3421 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     3481 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     3541 actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc ataatcagcc
     3601 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     3661 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     3721 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     3781 gttgtggttt gtccaaactc atcaatgtat cttagtcgac cgatgccctt gagagccttc
     3841 aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact
     3901 gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctcttccg cttcctcgct
     3961 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4021 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4081 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4141 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4201 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4261 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4321 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4381 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4441 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4501 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4561 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4621 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4681 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4741 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4801 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4861 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4921 gatctgt
//
