LOCUS       sequence-341543-        5247 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses C9-1 gRNA-12xMBS from hU6 promoter .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5247)
  AUTHORS   Chaudhary N, Nho SH, Cho H, Gantumur N, Ra JS, Myung K, Kim H
  TITLE     Background-suppressed live visualization of genomic loci with an 
            improved CRISPR system based on a split fluorophore.
  JOURNAL   Genome Res. 2020 Sep;30(9):1306-1316. doi: 10.1101/gr.260018.119. 
            Epub 2020 Sep 4.
  PUBMED    32887690
REFERENCE   2  (bases 1 to 5247)
  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"; doi: 
            "10.1101/gr.260018.119"; journalName: "Genome Res"; date: "2020-09";
            volume: "30"; issue: "9"; pages: "1306-1316"
COMMENT     Sequence Label: pM378
FEATURES             Location/Qualifiers
     source          1..5247
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..320
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     72..92
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     243..262
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        364..382
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        393..411
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        451..469
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        669..687
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_feature    766..883
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        932..1442
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1361..1379
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1464..2063
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1464..1483)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1960..1980
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2210..2331
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2221..2240)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2275..2294
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2579..2596)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2750..3338)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2830..2849)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3509..4300,4301..4369))
                     /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     4132..4151
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4370..4474)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4501..4956
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4588..4607)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4798..4819
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5087..5135
                     /note="synthetic polyadenylation signal"
     misc_feature    5149..5240
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5189..5208
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
       61 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      301 tgtggaaagg acgaaacacc gtggaatgga atggaatgga agtttgagag ctaggccggg
      361 agcacatgag gatcacccat gtgccacgag cgacatgagg atcacccatg tcgctcgtgt
      421 tccctcttcg tagaggatca ccctacgtct acatgaggat cacccatgtt ctggtagagg
      481 atcaccctac ctctcgtaga ggatcaccct acgtctgcag gaggatcacc cctgctctcg
      541 aggaggatca cccctcgtct gactgaggat cacccagtct ctgctagagg atcaccctag
      601 ctctccttga ggatcaccca aggggcctag caagttcaaa taaggctagt ccgttatcaa
      661 cttggccaac atgaggatca cccatgtctg cagggccaag tggcaccgag tcggtgcttt
      721 ttttgaattc tcgacctcga gacaaatggc agtattcatc cacaatttta aaagaaaagg
      781 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
      841 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
      901 cagcagagat ccactttggc cgcggctcga gggggttggg gttgcgcctt ttccaaggca
      961 gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg
     1021 ccgaccctgg gactcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg
     1081 ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct
     1141 tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact agtaccctcg
     1201 cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata
     1261 gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg
     1321 ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc
     1381 ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc
     1441 agggggatcc accggagctt accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     1501 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     1561 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     1621 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     1681 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     1741 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     1801 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
     1861 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     1921 gcgccggggt gcccgccttc ctggaaacct ccgcgccccg caacctcccc ttctacgagc
     1981 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     2041 tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga
     2101 gcgcacgacc ccatgcatcg gtacctttaa gaccaatgac ttacaaggca gctgtagatc
     2161 ttagccactt tctagagtcg gggcggccgg ccgcttcgag cagacatgat aagatacatt
     2221 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     2281 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     2341 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag
     2401 taaaacctct acaaatgtgg taaaatcgat aaggatccgt cgaccgatgc ccttgagagc
     2461 cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat
     2521 gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct tccgcttcct
     2581 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2641 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2701 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2761 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2821 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2881 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2941 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3001 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3061 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3121 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3181 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3241 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3301 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3361 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3421 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3481 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3541 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3601 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcgg gacccacgct
     3661 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3721 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3781 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3841 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3901 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3961 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4021 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4081 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4141 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4201 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4261 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4321 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4381 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4441 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4501 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4561 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4621 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4681 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     4741 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4801 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     4861 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     4921 acgcgaattt taacaaaata ttaacgctta caatttgcca ttcgccattc aggctgcgca
     4981 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagccc aagctaccat
     5041 gataagtaag taatattaag gtacgggagg tacttggagc ggccgcaata aaatatcttt
     5101 attttcatta catctgtgtg ttggtttttt gtgtgaatcg atagtactaa catacgctct
     5161 ccatcaaaac aaaacgaaac aaaacaaact agcaaaatag gctgtcccca gtgcaagtgc
     5221 aggtgccaga acatttctct atcgata
//
