LOCUS       sequence-388067-        4584 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Sortable sgRNA expression, Chen tracrRNA PMID: 24360272.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4584)
  TITLE     Schmierer unpublished plasmids
REFERENCE   2  (bases 1 to 4584)
  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"
COMMENT     Sequence Label: pCMV-BFP-U6-sgRNA
FEATURES             Location/Qualifiers
     source          1..4584
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             630..1331
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     promoter        1334..1582
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1334..1354
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1505..1524
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             1819..1839
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1848..1865
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1888..1905)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1894..2118
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2189..2205)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2189..2205)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2202..2224)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2213..2229
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2237..2243,2244..2261,2262..2267))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2282..2303
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2420..2437)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2591..3179)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2671..2690)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3350..4141,4142..4210))
                     /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     3973..3992
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4211..4315)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4394..4413)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gagatccgcg gccgccacca tgagcgagct gattaaggag aacatgcaca
      661 tgaagctcta catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg
      721 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc
      781 tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca
      841 accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg
      901 agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc
      961 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc
     1021 ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgaaacgctg taccccgctg
     1081 acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga
     1141 tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg
     1201 gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg
     1261 tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca
     1321 agcttaatta agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     1381 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     1441 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     1501 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     1561 cttgtggaaa ggacgaaaca ccggagacgg ttgtaaatga gcacacaaaa tacacatgct
     1621 aaaatattat attctatgac ctttataaaa attagaattt gcaaactttt aataaaaatg
     1681 agcaaaataa aaaaacgcta gtttttgtac gtctctgttt aagagctatg ctggaaacag
     1741 catagcaagt ttaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     1801 gctttttttg aattcgagcc caagaagaag aggaaagtct aaccggtcat catcaccatc
     1861 accattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     1921 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     1981 cctaataaaa tgagaaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2041 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2101 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg ataccgtcga
     2161 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     2221 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tagggtgcct
     2281 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2341 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2401 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     2461 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     2521 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2581 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2641 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2701 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2761 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     2821 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     2881 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     2941 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3001 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     3061 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     3121 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3181 aagatccttt gatcttttct acggggtctg acactcagtg gaacgaaaac tcacgttaag
     3241 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3301 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3361 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3421 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3481 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3541 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3601 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3661 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3721 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3781 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     3841 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     3901 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3961 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4021 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4081 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4141 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4201 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     4261 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     4321 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcgatctccc gatcccctag
     4381 ggtctactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     4441 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
     4501 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
     4561 atgtacgggc cagatatacg cgtt
//
