LOCUS       sequence-151422-        3389 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Encodes an sgRNA for spCas9 driven by hU6 promoter with a modified 
            scaffold (Chen et al. Cell 2013) and a spacer targeting the 3'UTR of
            ACTB mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3389)
  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 3389)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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) ACTB
FEATURES             Location/Qualifiers
     source          1..3389
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     240..260
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     297..313
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     297..313
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(442..690)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(500..519)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(670..690)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(775..791)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(776..792)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(816..835)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(817..835)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(845..862)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(845..861)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(854..876)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1003..1458
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1090..1109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1300..1321
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1484..1588
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1589..1657,1658..2449)
                     /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(1807..1826)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2620..3208
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3109..3128
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3362..3379
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
      241 caattaaccc tcactaaagg gaacaaaagc tggagctcca ccgcggtggc ggccgctcta
      301 gaactagtgg atcccccggg ctgcaggaaa aaaagcaccg actcggtgcc actttttcaa
      361 gttgataacg gactagcctt atttaaactt gctatgctgt ttccagcata gctcttaaac
      421 tggagcgagc atcccccaaa cggtgtttcg tcctttccac aagatatata aagccaagaa
      481 atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa
      541 aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt
      601 tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa
      661 atatgaagga atcatgggaa ataggccctc ttcctgcccg accttggtac cggatccagt
      721 cgactgaatt ggttccttta aagcctgctt ttttgtacac gatatcaagc ttatcgatac
      781 cgtcgacctc gagggggggc ccggtaccca attcgcccta tagtgagtcg tattacgcgc
      841 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
      901 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
      961 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     1021 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1081 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1141 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1201 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1261 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1321 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1381 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1441 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1501 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     1561 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     1621 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     1681 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1741 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     1801 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     1861 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1921 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1981 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     2041 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     2101 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     2161 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     2221 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     2281 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     2341 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     2401 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     2461 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     2521 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     2581 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2641 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2701 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2761 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2821 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2881 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2941 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3001 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3061 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3121 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3181 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3241 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3301 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3361 cagcgagtca gtgagcgagg aagcggaag
//
