LOCUS       sequence-370671-        3516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA plasmid encoding hU6 promoter driving an anti-safe targeting 
            sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3516)
  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 3516)
  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: phU6 ST
FEATURES             Location/Qualifiers
     source          1..3516
                     /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"
     promoter        696..944
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     696..716
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     867..886
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        964..1039
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1095..1115)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1095..1113)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1134..1150)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1134..1150)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1147..1169)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1158..1174
                     /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(1182..1188,1189..1206,1207..1212))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1227..1248
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1365..1382)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1536..2124)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1616..1635)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2201..2995)
                     /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(2201..2995)
                     /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(2312..2331)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2922..2941
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3030..3387)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3044..3179
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        3196..3387
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     3344..3364
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3389..3493)
                     /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 tgggtacccg
      661 tctcacaggc ggatcgatcc aaggtcgggc aggaagaggg cctatttccc atgattcctt
      721 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
      781 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      841 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      901 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggatgg tgacagttgt
      961 cgagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     1021 agtggcaccg agtcggtgct ttttttctcc gctgagcgta ctgagacgcc gcggtggagc
     1081 tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     1141 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     1201 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1261 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1321 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     1381 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     1441 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     1501 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     1561 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     1621 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     1681 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     1741 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     1801 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     1861 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     1921 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     1981 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2041 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2101 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2161 cagtggaacg aaaactcacg ttaagggatt ttggtcatga tcagaagaac tcgtcaagaa
     2221 ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc
     2281 ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct
     2341 gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt
     2401 ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg
     2461 gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt
     2521 ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat
     2581 gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg
     2641 catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc
     2701 ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag
     2761 ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcttgcagtt
     2821 cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca
     2881 gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata
     2941 gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa
     3001 acgatcctca tcctgtctct tgatcgatct ttgcaaaagc ctaggcctcc aaaaaagcct
     3061 cctcactact tctggaatag ctcagaggcc gaggcggcct cggcctctgc ataaataaaa
     3121 aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag gggcgggatg
     3181 ggcggagtta ggggcgggac tatggttgct gactaattga gatgcatgct ttgcatactt
     3241 ctgcctgctg gggagcctgg ggactttcca cacctggttg ctgactaatt gagatgcatg
     3301 ctttgcatac ttctgcctgc tggggagcct ggggactttc cacaccctaa ctgacacaca
     3361 ttccacagct ggttctttcc gcctcaggac tcttcctttt tcaatattat tgaagcattt
     3421 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3481 taggggttcc gcgcacattt ccccgaaaag tgccac
//
