LOCUS       sequence-495326-        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: InterIR gRNA #2
FEATURES             Location/Qualifiers
     source          1..4927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        272..347
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    399..516
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        565..1075
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     994..1012
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(1084..1086,1087..1089,1090..1803)
                     /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(1084..1086,1087..1089,1090..1800)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1129..1150)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1390..1409)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1737..1758
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1926..2047
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1963..1982)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2017..2036
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2188..2205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2359..2947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2439..2458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3118..3909,3910..3978))
                     /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     3741..3760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3979..4083)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4110..4565
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4197..4216)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4407..4428
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4696..4744
                     /note="synthetic polyadenylation signal"
     misc_feature    4758..4849
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4798..4817
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gtatatactt ttggttgtac tgttttagag ctagaaatag caagttaaaa
      301 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttaaagaa
      361 ttctcgacct cgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
      421 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
      481 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
      541 gatccacttt ggccgcggct cgagggggtt ggggttgcgc cttttccaag gcagccctgg
      601 gtttgcgcag ggacgcggct gctctgggcg tggttccggg aaacgcagcg gcgccgaccc
      661 tgggactcgc acattcttca cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct
      721 tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt
      781 cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc actagtaccc tcgcagacgg
      841 acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca atagcggctg
      901 ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg
      961 ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca agcctccgga
     1021 gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc cccaggggga
     1081 tccatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1141 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1201 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1261 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     1321 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1381 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1441 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1501 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     1561 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     1621 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1681 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     1741 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     1801 taaagcggcc gcgactctag atcataatca gccataccac atttgtagag gttttacttg
     1861 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     1921 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     1981 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     2041 tatcttagtc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg
     2101 cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga
     2161 caggtgccgg cagcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     2221 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     2281 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     2341 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2401 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2461 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2521 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     2581 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     2641 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2701 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     2761 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     2821 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     2881 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     2941 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3001 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3061 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     3121 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3181 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     3241 tgctgcaatg ataccgcggg acccacgctc accggctcca gatttatcag caataaacca
     3301 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3361 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3421 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     3481 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     3541 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     3601 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     3661 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3721 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     3781 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     3841 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     3901 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     3961 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4021 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     4081 gcgcacattt ccccgaaaag tgccacctga cgcgccctgt agcggcgcat taagcgcggc
     4141 gggtgtggtg gttacgcgca gcgtgaccgc tacaattgcc agcgccctag cgcccgctcc
     4201 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4261 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4321 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4381 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4441 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     4501 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     4561 aatttgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     4621 ttcgctatta cgccagccca agctaccatg ataagtaagt aatattaagg tacgggaggt
     4681 acttggagcg gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg
     4741 tgtgaatcga tagtactaac atacgctctc catcaaaaca aaacgaaaca aaacaaacta
     4801 gcaaaatagg ctgtccccag tgcaagtgca ggtgccagaa catttctcta tcgataggta
     4861 ccgattagtg aacggatctc gacggtatcg atcacgagac tagcctcgag cggccgcccc
     4921 cttcacc
//
