LOCUS       sequence-341548-        5248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  (Empty Backbone) Expresses gRNA-12xMBS from hU6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5248)
  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 5248)
  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: pM355
FEATURES             Location/Qualifiers
     source          1..5248
                     /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        365..383
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        394..412
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        452..470
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        670..688
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_feature    767..884
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        933..1443
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1362..1380
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1465..2064
                     /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(1465..1484)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1961..1981
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2211..2332
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2222..2241)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2276..2295
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2580..2597)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2751..3339)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2831..2850)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3510..4301,4302..4370))
                     /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     4133..4152
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4371..4475)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4502..4957
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4589..4608)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4799..4820
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5088..5136
                     /note="synthetic polyadenylation signal"
     misc_feature    5150..5241
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5190..5209
                     /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 gggagaccga gagagggtct cagtttgaga gctaggccgg
      361 gagcacatga ggatcaccca tgtgccacga gcgacatgag gatcacccat gtcgctcgtg
      421 ttccctcttc gtagaggatc accctacgtc tacatgagga tcacccatgt tctggtagag
      481 gatcacccta cctctcgtag aggatcaccc tacgtctgca ggaggatcac ccctgctctc
      541 gaggaggatc acccctcgtc tgactgagga tcacccagtc tctgctagag gatcacccta
      601 gctctccttg aggatcaccc aaggggccta gcaagttcaa ataaggctag tccgttatca
      661 acttggccaa catgaggatc acccatgtct gcagggccaa gtggcaccga gtcggtgctt
      721 tttttgaatt ctcgacctcg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
      781 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
      841 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
      901 acagcagaga tccactttgg ccgcggctcg agggggttgg ggttgcgcct tttccaaggc
      961 agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa acgcagcggc
     1021 gccgaccctg ggactcgcac attcttcacg tccgttcgca gcgtcacccg gatcttcgcc
     1081 gctacccttg tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc
     1141 ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg cacgtctcac tagtaccctc
     1201 gcagacggac agcgccaggg agcaatggca gcgcgccgac cgcgatgggc tgtggccaat
     1261 agcggctgct cagcagggcg cgccgagagc agcggccggg aaggggcggt gcgggaggcg
     1321 gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag
     1381 cctccggagc gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga cctctctccc
     1441 cagggggatc caccggagct taccatgacc gagtacaagc ccacggtgcg cctcgccacc
     1501 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     1561 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     1621 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     1681 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     1741 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     1801 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     1861 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     1921 cgcgccgggg tgcccgcctt cctggaaacc tccgcgcccc gcaacctccc cttctacgag
     1981 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     2041 atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg
     2101 agcgcacgac cccatgcatc ggtaccttta agaccaatga cttacaaggc agctgtagat
     2161 cttagccact ttctagagtc ggggcggccg gccgcttcga gcagacatga taagatacat
     2221 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     2281 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     2341 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
     2401 gtaaaacctc tacaaatgtg gtaaaatcga taaggatccg tcgaccgatg cccttgagag
     2461 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     2521 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc
     2581 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2641 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2701 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2761 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2821 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2881 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2941 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3001 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3061 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3121 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3181 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3241 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3301 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3361 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3421 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3481 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3541 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3601 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg ggacccacgc
     3661 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3721 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3781 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3841 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3901 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3961 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4021 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4081 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4141 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4201 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4261 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4321 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4381 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4441 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4501 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4561 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4621 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4681 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     4741 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4801 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4861 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4921 aacgcgaatt ttaacaaaat attaacgctt acaatttgcc attcgccatt caggctgcgc
     4981 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagcc caagctacca
     5041 tgataagtaa gtaatattaa ggtacgggag gtacttggag cggccgcaat aaaatatctt
     5101 tattttcatt acatctgtgt gttggttttt tgtgtgaatc gatagtacta acatacgctc
     5161 tccatcaaaa caaaacgaaa caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg
     5221 caggtgccag aacatttctc tatcgata
//
