LOCUS       sequence-152737-        3386 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes an sgRNA for spCas9 driven by hU6 promoter with a modified 
            scaffold (Chen et al. Cell 2013) and BbsI sites that allow insertion
            of a spacer sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3386)
  AUTHORS   Nelles DA, Fang MY, O'Connell MR, Xu JL, Markmiller SJ, Doudna JA, 
            Yeo GW
  TITLE     Programmable RNA Tracking in Live Cells with CRISPR/Cas9.
  JOURNAL   Cell. 2016 Mar 16. pii: S0092-8674(16)30204-5. doi: 
            10.1016/j.cell.2016.02.054.
  PUBMED    26997482
REFERENCE   2  (bases 1 to 3386)
  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: "Cell. 2016 
            Mar 16. pii: S0092-8674(16)30204-5. doi: 
            10.1016/j.cell.2016.02.054."
COMMENT     Sequence Label: pBluescriptSKII+ U6-sgRNA(F+E) empty
FEATURES             Location/Qualifiers
     source          1..3386
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     669..685
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        771..1019
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     771..791
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     942..961
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1145..1161)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1145..1161)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1198..1218)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1198..1216)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1237..1253)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1237..1253)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1250..1272)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1261..1277
                     /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(1285..1291,1292..1309,1310..1315))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1330..1351
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1468..1485)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1639..2227)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1719..1738)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2398..3189,3190..3258))
                     /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     3021..3040
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3259..3363)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttgatatc gtgtacaaaa aagcaggctt
      721 taaaggaacc aattcagtcg actggatccg gtaccaaggt cgggcaggaa gagggcctat
      781 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa
      841 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      901 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      961 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     1021 ggtcttcgag aagacctgtt taagagctat gctggaaaca gcatagcaag tttaaataag
     1081 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt cctgcagccc
     1141 gggggatcca ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct
     1201 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     1261 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     1321 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     1381 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     1441 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     1501 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     1561 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     1621 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     1681 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     1741 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     1801 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     1861 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     1921 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     1981 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     2041 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     2101 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     2161 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     2221 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     2281 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     2341 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     2401 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     2461 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     2521 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     2581 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     2641 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     2701 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     2761 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     2821 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     2881 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     2941 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3001 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     3061 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     3121 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     3181 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     3241 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     3301 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     3361 gcgcacattt ccccgaaaag tgccac
//
