LOCUS       sequence-370668-        3545 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA plasmid encoding mU6 promoter driving an anti-safe targeting 
            sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3545)
  AUTHORS   Brewer WJ, Xet-Mull AM, Yu A, Sweeney MI, Walton EM, Tobin DM
  TITLE     Macrophage NFATC2 mediates angiogenic signaling during mycobacterial
            infection.
  JOURNAL   Cell Rep. 2022 Dec 13;41(11):111817. doi: 
            10.1016/j.celrep.2022.111817.
  PUBMED    36516756
REFERENCE   2  (bases 1 to 3545)
  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 Rep.";
            date: "2022-12-13"; volume: "41(11):111817. doi"; pages: " 
            10.1016/j.celrep.2022.111817"
COMMENT     Sequence Label: pmU6 ST
FEATURES             Location/Qualifiers
     source          1..3545
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..314
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     233..253
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        335..410
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(455..475)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(455..473)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(494..510)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(494..510)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(507..529)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    518..534
                     /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(542..548,549..566,567..572))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    587..608
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(725..742)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(896..1484)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(976..995)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1561..2355)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(1561..2355)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1672..1691)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2282..2301
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2390..2747)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2404..2539
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        2556..2747
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     2704..2724
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(2749..2853)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2879..3334)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3016..3037)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3228..3247
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3461..3483
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3475..3492
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3476..3492
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3502..3521
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3502..3520
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gatccgacgc cgccatctct aggcccgcgc cggccccctc gcacagactt gtgggagaag
       61 ctcggctact cccctgcccc ggttaatttg catataatat ttcctagtaa ctatagaggc
      121 ttaatgtgcg ataaaagaca gataatctgt tctttttaat actagctaca ttttacatga
      181 taggcttgga tttctataag agatacaaat actaaattat tattttaaaa aacagcacaa
      241 aaggaaactc accctaactg taaagtaatt gtgtgttttg agactataaa tatcccttgg
      301 agaaaagcct tgtttggcta agtactctaa cagggtttta gagctagaaa tagcaagtta
      361 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttgta
      421 ctgagacgcc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct
      481 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
      541 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
      601 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
      661 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
      721 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
      781 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
      841 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
      901 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
      961 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     1021 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     1081 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     1141 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     1201 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     1261 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     1321 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     1381 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     1441 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     1501 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     1561 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
     1621 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
     1681 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
     1741 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
     1801 gacgagatcc tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc
     1861 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
     1921 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
     1981 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
     2041 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
     2101 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
     2161 cgctgcctcg tcttgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
     2221 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
     2281 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
     2341 atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcgatct ttgcaaaagc
     2401 ctaggcctcc aaaaaagcct cctcactact tctggaatag ctcagaggcc gaggcggcct
     2461 cggcctctgc ataaataaaa aaaattagtc agccatgggg cggagaatgg gcggaactgg
     2521 gcggagttag gggcgggatg ggcggagtta ggggcgggac tatggttgct gactaattga
     2581 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca cacctggttg
     2641 ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc
     2701 cacaccctaa ctgacacaca ttccacagct ggttctttcc gcctcaggac tcttcctttt
     2761 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     2821 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     2881 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     2941 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     3001 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     3061 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     3121 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     3181 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     3241 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     3301 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac
     3361 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     3421 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     3481 acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg tacccgtctc
     3541 agata
//
