LOCUS       sequence-242297-        4407 bp    DNA     circular SYN 14-APR-2025
DEFINITION  U6 driven NmCas9 sgRNA expression vector for cloning own guides.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4407)
  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 4407)
  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: "Nat 
            Commun."; date: "2018-11-19"; volume: "9(1):4856. doi"; pages: " 
            10.1038/s41467-018-07310-x"
COMMENT     Sequence Label: NmCas9_sgRNA_expression_in_pBluescript
FEATURES             Location/Qualifiers
     source          1..4407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(32..37,38..55,56..62)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    70..86
                     /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     75..97
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     94..110
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     94..110
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     129..149
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        131..149
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        196..444
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     196..216
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     367..386
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(575..1369)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(686..705)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1296..1315
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_RNA        1562..1654
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     primer_bind     complement(1682..1698)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1683..1699)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1723..1742)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1724..1742)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1752..1769)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1752..1768)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1761..1783)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1910..2365
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1997..2016)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2207..2228
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2391..2495
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2496..2564,2565..3356)
                     /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(2714..2733)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3527..4115
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4016..4035
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4269..4286
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    join(4403..4407,1..17)
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
       61 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
      121 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg
      181 gccgccccct tcaccgaggg cctatttccc atgattcctt catatttgca tatacgatac
      241 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
      301 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
      361 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
      421 atcttgtgga aaggacgaaa caccggtgtg caggtgagtg atccaaacgc ccggcggcaa
      481 ccgagcgttc tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg
      541 agtccaagcg agctctcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat
      601 agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag
      661 cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc
      721 ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca
      781 tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc
      841 gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat
      901 catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg
      961 cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag
     1021 ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca
     1081 cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc
     1141 aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca
     1201 gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga
     1261 acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct
     1321 ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc
     1381 ctcatcctgt ctcttgatca gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga
     1441 aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca
     1501 attccgacgg atcagctagc agaacctgct gacgttgtag ctccctttct catttcggaa
     1561 acgaaatgag aaccgttgct acaataaggc cgtctgaaaa gatgtgccgc aacgctctgc
     1621 cccttaaagc ttctgcttta aggggcatcg tttatttttt tgaattcgat atcaagctta
     1681 tcgataccgt cgacctcgag ggggggcccg gtacccaatt cgccctatag tgagtcgtat
     1741 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     1801 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     1861 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     1921 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     1981 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2041 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2101 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     2161 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2221 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     2281 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     2341 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     2401 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2461 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     2521 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     2581 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     2641 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     2701 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     2761 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     2821 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     2881 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     2941 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3001 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     3061 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3121 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3181 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     3241 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     3301 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     3361 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3421 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3481 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3541 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3601 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3661 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3721 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     3781 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3841 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3901 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3961 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4021 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4081 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4141 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4201 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4261 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     4321 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     4381 cgggcagtga gcgcaacgca attaatg
//
