LOCUS       sequence-135959-        3396 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA scaffold and human U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3396)
  AUTHORS   Vidigal JA, Ventura A
  TITLE     Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 
            libraries.
  JOURNAL   Nat Commun. 2015 Aug 17;6:8083. doi: 10.1038/ncomms9083.
  PUBMED    26278926
REFERENCE   2  (bases 1 to 3396)
  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: "Nat 
            Commun."; date: "2015-08-17"; volume: "6:8083. doi"; pages: " 
            10.1038/ncomms9083"
COMMENT     Sequence Label: pDonor_hU6
FEATURES             Location/Qualifiers
     source          1..3396
                     /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     677..693
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     677..693
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_RNA        724..799
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        890..1138
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     890..910
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1061..1080
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1162..1178)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1163..1179)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1208..1228)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1208..1226)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1247..1263)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1247..1263)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1260..1282)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1271..1287
                     /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(1295..1301,1302..1319,1320..1325))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1340..1361
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1478..1495)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1649..2237)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1729..1748)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2408..3199,3200..3268))
                     /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     3031..3050
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3269..3373)
                     /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 tggagctcca
      661 ccgcggtggc ggccgctcta gaactagtgg atcccccggg ctgcaggaat tcgatgaaga
      721 cctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
      781 agtggcaccg agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc
      841 tagtccgttt ttagcgcgtg cgccaattct gcagacaaat ggctctagag agggcctatt
      901 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
      961 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     1021 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     1081 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgt
     1141 ttgggtcttc atcaagctta tcgataccgt cgacctcgag ggggggcccg gtacccagct
     1201 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc
     1261 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     1321 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     1381 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     1441 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     1501 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     1561 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     1621 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     1681 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     1741 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     1801 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     1861 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     1921 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     1981 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2041 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     2101 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2161 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2221 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2281 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2341 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2401 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2461 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2521 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     2581 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     2641 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     2701 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     2761 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     2821 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     2881 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     2941 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3001 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3061 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3121 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3181 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3241 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3301 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3361 taggggttcc gcgcacattt ccccgaaaag tgccac
//
