LOCUS       sequence-204778-        3342 bp    DNA     circular SYN 15-APR-2025
DEFINITION  U6 driven NmCas9 sgRNA expression for VEGFA site 3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3342)
  AUTHORS   Bolukbasi MF, Liu P, Luk K, Kwok SF, Gupta A, Amrani N, Sontheimer 
            EJ, Zhu LJ, Wolfe SA
  TITLE     Orthogonal Cas9-Cas9 chimeras provide a versatile platform for 
            genome editing.
  JOURNAL   Nat Commun. 2018 Nov 19;9(1):4856. doi: 10.1038/s41467-018-07310-x.
  PUBMED    30451839
REFERENCE   2  (bases 1 to 3342)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2018-11-19"; volume: "9(1):4856. doi"; pages: " 
            10.1038/s41467-018-07310-x"
COMMENT     Sequence Label: pBluescript_VEGFA-TS3_NmCas9
FEATURES             Location/Qualifiers
     source          1..3342
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        20..124
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(125..193,194..985)
                     /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(343..362)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1156..1744
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1645..1664
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1898..1915
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2032..2053
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2068..2073,2074..2091,2092..2098)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2106..2122
                     /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     2111..2133
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2130..2146
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2130..2146
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2165..2185
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2167..2185
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2232..2480
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2232..2252
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2403..2422
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2533..2625
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     primer_bind     complement(2653..2669)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2654..2670)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2694..2713)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2695..2713)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2723..2740)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2723..2739)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2732..2754)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2881..3336
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2968..2987)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3178..3199
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
       61 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
      121 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
      181 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
      241 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
      301 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
      361 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
      421 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
      481 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
      541 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
      601 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
      661 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
      721 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
      781 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
      841 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
      901 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
      961 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     1021 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1081 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1141 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1201 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1261 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     1321 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1381 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1441 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1501 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1561 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1621 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1681 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1741 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     1801 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     1861 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     1921 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     1981 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     2041 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     2101 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     2161 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctccaccgcg gtggcggccg
     2221 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     2281 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     2341 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     2401 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     2461 tgtggaaagg acgaaacacc gcggggagaa ggccaggggt cactgttgta gctccctttc
     2521 tcatttcgga aacgaaatga gaaccgttgc tacaataagg ccgtctgaaa agatgtgccg
     2581 caacgctctg ccccttaaag cttctgcttt aaggggcatc gtttattttt ttgaattcga
     2641 tatcaagctt atcgataccg tcgacctcga gggggggccc ggtacccaat tcgccctata
     2701 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     2761 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     2821 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg
     2881 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2941 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3001 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3061 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3121 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3181 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3241 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3301 acgcgaattt taacaaaata ttaacgctta caatttaggt gg
//
