LOCUS       sequence-68838-e        4301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid contains expression cassette for sgRNA targeting 
            tdTomato..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4301)
  AUTHORS   Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, 
            Thomson JA
  TITLE     Efficient genome engineering in human pluripotent stem cells using 
            Cas9 from Neisseria meningitidis.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Aug 12.
  PUBMED    23940360
REFERENCE   2  (bases 1 to 4301)
  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: "Proc Natl 
            Acad Sci U S A. 2013 Aug 12."
COMMENT     Sequence Label: pSTBlue-U6-sgRNA(tdTomato)
FEATURES             Location/Qualifiers
     source          1..4301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        143..391
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     143..163
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     314..333
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        444..536
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(634..652)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(635..652)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(666..683)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(666..682)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(675..697)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(823..1278)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(960..981)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1172..1191
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1305..1409
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1410..1478,1479..2270)
                     /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     complement(1628..1647)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(2405..2424)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2416..3231
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2416..3231
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2489..2508)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3316..3904
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3805..3824
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4058..4075
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4192..4213
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4228..4233,4234..4251,4252..4258)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4266..4282
                     /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)."
     primer_bind     4271..4293
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(4290..4301,1..5)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(4290..4301,1..5)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atgaccatga ttacgccaag ctctaatacg actcactata gggaaagctc ggtaccacgc
       61 atgctgcaga cgcgttacgt atcggatcca gaattcgtga tccaagctcg gatccgatat
      121 cgacgtcaag gtcgggcagg aagagggcct atttcccatg attccttcat atttgcatat
      181 acgatacaag gctgttagag agataattag aattaatttg actgtaaaca caaagatatt
      241 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      301 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      361 tttatatatc ttgtggaaag gacgaaacac cgtacgtgaa gcaccccgcc gacatgttgt
      421 agctcccttt ctcatttcgg aaacgaaatg agaaccgttg ctacaataag gccgtctgaa
      481 aagatgtgcc gcaacgctct gccccttaaa gcttctgctt taaggggcat cgtttatttc
      541 ggtttttttg catctgaatt cgtcgacaag cttctcgagc ctaggctagc tctagaccac
      601 acgtgtgggg gcccgagctc gcggccgctg tattctatag tgtcacctaa atggccgcac
      661 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
      721 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
      781 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggaaattgta agcgttaata
      841 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
      901 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
      961 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     1021 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     1081 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     1141 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     1201 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     1261 cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1321 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1381 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1441 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1501 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1561 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1621 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1681 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1741 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1801 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     1861 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     1921 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     1981 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2041 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     2101 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     2161 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2221 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2281 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2341 aggtgaagat cctttttgat aatctcatga acaataaaac tgtctgctta cataaacagt
     2401 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta
     2461 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     2521 tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa
     2581 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     2641 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     2701 ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat
     2761 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     2821 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     2881 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     2941 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     3001 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     3061 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     3121 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     3181 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta agaattaatt
     3241 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3301 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3361 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3421 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     3481 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3541 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3601 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3661 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3721 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3781 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3841 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3901 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3961 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4021 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     4081 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     4141 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     4201 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     4261 tggaattgtg agcggataac aatttcacac aggaaacagc t
//
