LOCUS       sequence-268971-        8730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a guide RNA (gRNA) to target human IRF-1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8730)
  AUTHORS   Fujita T, Fujii H
  TITLE     Identification of Proteins Associated with an IFNgamma-Responsive 
            Promoter by a Retroviral Expression System for enChIP Using CRISPR.
  JOURNAL   PLoS One. 2014 Jul 22;9(7):e103084. doi: 
            10.1371/journal.pone.0103084. eCollection 2014.
  PUBMED    25051498
REFERENCE   2  (bases 1 to 8730)
  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: "PLoS One.";
            date: "2014-07-22"; volume: "9(7):e103084. doi"; pages: " 
            10.1371/journal.pone.0103084. eCollection 2014"
COMMENT     Sequence Label: gRNA-hIRF-1 #12/pSIR
FEATURES             Location/Qualifiers
     source          1..8730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             121..590
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    655..1012
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1079..1495
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1425..1447
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1463..1479
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        2129..2377
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2129..2149
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2300..2319
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2398..2473
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(2485..2509)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3468..4262
                     /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(3522..3541)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4132..4151
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5714..5730)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5714..5730)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5727..5749)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5738..5754
                     /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(5762..5768,5769..5786,5787..5792))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5807..5828
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5945..5962)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6116..6704)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6196..6215)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6875..7666,7667..7735))
                     /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     7498..7517
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7736..7840)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7908..7926
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7964..7986)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8086..8105
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8299..8321
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8313..8330
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8314..8330
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaaatac ataactgaga atagaaaagt tcagatcaag gtcaggaaca gatggaacag
      121 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
      181 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
      241 ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag
      301 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
      361 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
      421 aaaagagccc acaacccctc actcggcgcg ccagtcctcc gattgactga gtcgcccggg
      481 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
      541 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttgggggct
      601 cgtccgggat cgggagaccc ctgcccaggg accaccgacc caccaccggg aggtaagctg
      661 gccagcaact tatctgtgtc tgtccgattg tctagtgtct atgactgatt ttatgcgcct
      721 gcgtcggtac tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga
      781 gttcggaaca cccggccgca accctgggag acgtcccagg gacttcgggg gccgtttttg
      841 tggcccgacc tgagtccaaa aatcccgatc gttttggact ctttggtgca ccccccttag
      901 aggagggata tgtggttctg gtaggagacg agaacctaaa acagttcccg cctccgtctg
      961 aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg tcttgtctgc tgcagcatcg
     1021 ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc tgaaaatatg ggcccgcggg
     1081 ccagactgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat
     1141 cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga
     1201 atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac
     1261 ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtccccta
     1321 catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca
     1381 ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg
     1441 ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgccaaacc
     1501 taaacctcaa gttctttctg acagtggggg gccgctcatc gacctactta cagaagaccc
     1561 cccgccttat agggacccaa gaccaccccc ttccgacagg gacggaaatg gtggagaagc
     1621 gacccctgcg ggagaggcac cggacccctc cccaatggca tctcgcctac gtgggagacg
     1681 ggagccccct gtggccgact ccactacctc gcaggcattc cccctccgcg caggaggaaa
     1741 cggacagctt caatactggc cgttctcctc ttctgacctt tacaactgga aaaataataa
     1801 cccttctttt tctgaagatc caggtaaact gacagctctg atcgagtctg tcctcatcac
     1861 ccatcagccc acctgggacg actgtcagca gctgttgggg actctgctga ccggagaaga
     1921 aaaacaacgg gtgctcttag aggctagaaa ggcggtgcgg ggcgatgatg ggcgccccac
     1981 tcaactgccc aatgaagtcg atgccgcttt tcccctcgag cggccgccag tgtgatggat
     2041 atctgcagaa ttcgcccttt gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac
     2101 tggatccggt accaaggtcg ggcaggaaga gggcctattt cccatgattc cttcatattt
     2161 gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa
     2221 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     2281 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     2341 cttggcttta tatatcttgt ggaaaggacg aaacaccgcg ggggcgctgg gctgtccgtt
     2401 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     2461 accgagtcgg tgcttttttt ctagacccag ctttcttgta caaagttggc attaaagggc
     2521 gaattccagc acactggcgg ccgttactag tggatccgag ctcggtacca agcttgtcga
     2581 cggatccgaa ttcgtcgagg gaagaaataa taaagacatt aaaatcactt ttgtaaactt
     2641 tttaaataag ggaaattgat taaaacaacg cgaagttgta aaatgccata tactgcatac
     2701 attttgtggg aaaaaatgtg catttacctg ctcaccaaat tatcttatta gctagcagta
     2761 gagctctttg tgtgatggga agatgggata acagctgttg ttactgtact ggtttttttt
     2821 tttaatatat ttaaaaaaaa gagtgtctcc taaagtgtaa gcgctatcat gctggactaa
     2881 tgttttaatt taatttaatt gttgtcttgg tagagatggg gtctcgctat tttgctcacg
     2941 cagatctcaa actcctgacg cccaggtaca ctcccatctc ggcctcctaa aatggtggga
     3001 tcacagacgt cagccaccga gcccggtcac ttttttgtat tccccacagt attgatgtat
     3061 atcttctgcg ttcaaaagca attttttaaa gcctcataac gtggtaacag aatactttgc
     3121 acattacaaa attcagaaca cggaaacaag aagctcgctt ttttttcccc cctatttcgg
     3181 tttggccctt tagatttccc ctcccccacc ggggcgggac ttcccgccga cttctttcag
     3241 gttctcagtt cggtccgcca actgtcgtat aaaggcgctg cctcaggcca gaggcctcac
     3301 aaagcgcgtc gagacagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt
     3361 tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct gatggcgcag
     3421 gggatcaaga tcgatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3481 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3541 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3601 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     3661 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3721 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3781 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3841 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3901 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     3961 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     4021 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4081 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4141 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4201 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4261 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4321 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4381 cggctggatg atcctccagc gcggggatct catgctggag atcccgcgat tagtccaatt
     4441 tgttaaagac aggatatcag tggtccaggc tctagttttg actcaacaat atcaccagct
     4501 gaagcctata gagtacgagc catagataaa ataaaagatt ttatttagtc tccagaaaaa
     4561 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg
     4621 caaggcatgg aaaaatacat aactgagaat agaaaagttc agatcaaggt caggaacaga
     4681 tggaacaggg tcgaccggtc gaccggtcga ccctagagaa ccatcagatg tttccagggt
     4741 gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc
     4801 gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc
     4861 ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca ataaaccctc
     4921 ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt
     4981 gactacccgt cagcgggggt ctttcacatg cagcatgtat caaaattaat ttggtttttt
     5041 ttcttaagta tttacattaa atggccatag tacttaaagt tacattggct tccttgaaat
     5101 aaacatggag tattcagaat gtgtcataaa tatttctaat tttaagatag tatctccatt
     5161 ggctttctac tttttctttt attttttttt gtcctctgtc ttccatttgt tgttgttgtt
     5221 gtttgtttgt ttgtttgttg gttggttggt taattttttt ttaaagatcc tacactatag
     5281 ttcaagctag actattagct actctgtaac ccagggtgac cttgaagtca tgggtagcct
     5341 gctgttttag ccttcccaca tctaagatta caggtatgag ctatcatttt tggtatattg
     5401 attgattgat tgattgatgt gtgtgtgtgt gattgtgttt gtgtgtgtga ttgtgtatat
     5461 gtgtgtatgg ttgtgtgtga ttgtgtgtat gtatgtttgt gtgtgattgt gtgtgtgtga
     5521 ttgtgcatgt gtgtgtgtgt gattgtgttt atgtgtatga ttgtgtgtgt gtgtgtgtgt
     5581 gtgtgtgtgt gtgtgtgtgt gtgtgtgttg tgtatatata tttatggtag tgagaggcaa
     5641 cgctccggct caggtgtcag gttggttttt gagacagagt ctttcactta gcttggaatt
     5701 aattcgtaat catgtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5761 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5821 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5881 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5941 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     6001 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     6061 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6121 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6181 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6241 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6301 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6361 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6421 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6481 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6541 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6601 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6661 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6721 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6781 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6841 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6901 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6961 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     7021 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     7081 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7141 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7201 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7261 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7321 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7381 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7441 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7501 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7561 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7621 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7681 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7741 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7801 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7861 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     7921 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     7981 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     8041 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     8101 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     8161 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     8221 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     8281 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gccaagctag
     8341 ctttgctctt aggagtttcc taatacatcc caaactcaaa tatataaagc atttgacttg
     8401 ttctatgccc tagggggcgg ggggaagcta agccagcttt ttttaacatt taaaatgtta
     8461 attccatttt aaatgcacag atgtttttat ttcataaggg tttcaatgtg catgaatgct
     8521 gcaatattcc tgttaccaaa gctagtataa ataaaaatag ataaacgtgg aaattactta
     8581 gagtttctgt cattaacgtt tccttcctca gttgacaaca taaatgcgct gctgagaagc
     8641 cagtttgcat ctgtcaggat caatttccca ttatgccagt catattaatt actagtcaat
     8701 tagttgattt ttatttttga catatacatg
//
