LOCUS       sequence-101745-        8634 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 8634)
  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 8634)
  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..8634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    580..937
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             996..1412
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1342..1364
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1380..1396
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        2046..2294
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2046..2066
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2217..2236
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2315..2390
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(2402..2426)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3385..4179
                     /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(3439..3458)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4049..4068
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5618..5634)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5618..5634)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5631..5653)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5642..5658
                     /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(5666..5672,5673..5690,5691..5696))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5711..5732
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5849..5866)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6020..6608)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6100..6119)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6779..7570,7571..7639))
                     /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     7402..7421
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7640..7744)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7812..7830
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7868..7890)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7990..8009
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8203..8225
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8217..8234
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8218..8234
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca gatggaacag
      121 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
      181 gaacagatgg tccccagatg cggtccagcc ctcagcagtt tctagagaac catcagatgt
      241 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
      301 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
      361 ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa
      421 taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc
      481 tgagtgattg actacccgtc agcgggggtc tttcatttgg gggctcgtcc gggatcggga
      541 gacccctgcc cagggaccac cgacccacca ccgggaggta agctggccag caacttatct
      601 gtgtctgtcc gattgtctag tgtctatgac tgattttatg cgcctgcgtc ggtactagtt
      661 agctaactag ctctgtatct ggcggacccg tggtggaact gacgagttcg gaacacccgg
      721 ccgcaaccct gggagacgtc ccagggactt cgggggccgt ttttgtggcc cgacctgagt
      781 ccaaaaatcc cgatcgtttt ggactctttg gtgcaccccc cttagaggag ggatatgtgg
      841 ttctggtagg agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg
      901 tttgggaccg aagccgcgcc gcgcgtcttg tctgctgcag catcgttctg tgttgtctct
      961 gtctgactgt gtttctgtat ttgtctgaga atatgggcca gactgttacc actcccttaa
     1021 gtttgacctt aggtcactgg aaagatgtcg agcggatcgc tcacaaccag tcggtagatg
     1081 tcaagaagag acgttgggtt accttctgct ctgcagaatg gccaaccttt aacgtcggat
     1141 ggccgcgaga cggcaccttt aaccgagacc tcatcaccca ggttaagatc aaggtctttt
     1201 cacctggccc gcatggacac ccagaccagg tcccctacat cgtgacctgg gaagccttgg
     1261 cttttgaccc ccctccctgg gtcaagccct ttgtacaccc taagcctccg cctcctcttc
     1321 ctccatccgc cccgtctctc ccccttgaac ctcctcgttc gaccccgcct cgatcctccc
     1381 tttatccagc cctcactcct tctctaggcg ccaaacctaa acctcaagtt ctttctgaca
     1441 gtggggggcc gctcatcgac ctacttacag aagacccccc gccttatagg gacccaagac
     1501 cacccccttc cgacagggac ggaaatggtg gagaagcgac ccctgcggga gaggcaccgg
     1561 acccctcccc aatggcatct cgcctacgtg ggagacggga gccccctgtg gccgactcca
     1621 ctacctcgca ggcattcccc ctccgcgcag gaggaaacgg acagcttcaa tactggccgt
     1681 tctcctcttc tgacctttac aactggaaaa ataataaccc ttctttttct gaagatccag
     1741 gtaaactgac agctctgatc gagtctgttc tcatcaccca tcagcccacc tgggacgact
     1801 gtcagcagct gttggggact ctgctgaccg gagaagaaaa acaacgggtg ctcttagagg
     1861 ctagaaaggc ggtgcggggc gatgatgggc gccccactca actgcccaat gaagtcgatg
     1921 ccgcttttcc cctcgagcgg ccgccagtgt gatggatatc tgcagaattc gccctttgta
     1981 caaaaaagca ggctttaaag gaaccaattc agtcgactgg atccggtacc aaggtcgggc
     2041 aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     2101 gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     2161 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     2221 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     2281 aaggacgaaa caccgcgggg gcgctgggct gtccgtttta gagctagaaa tagcaagtta
     2341 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttcta
     2401 gacccagctt tcttgtacaa agttggcatt aaagggcgaa ttccagcaca ctggcggccg
     2461 ttactagtgg atccgagctc ggtaccaagc ttgtcgacgg atccgaattc gtcgagggaa
     2521 gaaataataa agacattaaa atcacttttg taaacttttt aaataaggga aattgattaa
     2581 aacaacgcga agttgtaaaa tgccatatac tgcatacatt ttgtgggaaa aaatgtgcat
     2641 ttacctgctc accaaattat cttattagct agcagtagag ctctttgtgt gatgggaaga
     2701 tgggataaca gctgttgtta ctgtactggt tttttttttt aatatattta aaaaaaagag
     2761 tgtctcctaa agtgtaagcg ctatcatgct ggactaatgt tttaatttaa tttaattgtt
     2821 gtcttggtag agatggggtc tcgctatttt gctcacgcag atctcaaact cctgacgccc
     2881 aggtacactc ccatctcggc ctcctaaaat ggtgggatca cagacgtcag ccaccgagcc
     2941 cggtcacttt tttgtattcc ccacagtatt gatgtatatc ttctgcgttc aaaagcaatt
     3001 ttttaaagcc tcataacgtg gtaacagaat actttgcaca ttacaaaatt cagaacacgg
     3061 aaacaagaag ctcgcttttt tttcccccct atttcggttt ggccctttag atttcccctc
     3121 ccccaccggg gcgggacttc ccgccgactt ctttcaggtt ctcagttcgg tccgccaact
     3181 gtcgtataaa ggcgctgcct caggccagag gcctcacaaa gcgcgtcgag acagcaagcg
     3241 aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact
     3301 ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagatcg atctgatcaa
     3361 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3421 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3481 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3541 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     3601 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3661 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     3721 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     3781 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     3841 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     3901 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     3961 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4021 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4081 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4141 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     4201 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     4261 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4321 gggatctcat gctggagatc ccgcgattag tccaatttgt taaagacagg atatcagtgg
     4381 tccaggctct agttttgact caacaatatc accagctgaa gcctatagag tacgagccat
     4441 agataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct
     4501 gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac
     4561 tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacaggtcga cctagagaac
     4621 catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta
     4681 accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag
     4741 agcccacaac ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc
     4801 gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga
     4861 gggtctcctc tgagtgattg actacccgtc agcgggggtc tcttcacatg cagcatgtat
     4921 caaaattaat ttggtttttt tttcttaagt atttacatta aatggcccat agtacttaaa
     4981 gtaacatggg cttccttgaa ataaacatgg agtattcaga atgtgtcata aatatttcta
     5041 attttaagat agtatctcca ttggctatct actttttctt atattttttt ttgtcctctg
     5101 tcttccattc gttgttgttg ttgtttgttt gtttgtttgt tggttggttg gttaattttt
     5161 ttttaaagat cctacactat agttcaagct agactattag ctactctgta acccagggtg
     5221 acctttgaag tcatgggtag cctgctgttt tagccttccc acatctaaga ttacaggtat
     5281 gagctatcat ttttggtata ttgattgatt gattgattga tgtgtgtgtg tgtgattgtg
     5341 tttgtgtgtg tgattgtgta tatgtgtgta tggttgtgtg tgattgtgtg tatgtatgtt
     5401 tgtgtgtgat tgtgtgtgtg tgattgtgca tgtgtgtgtg tgtgattgtg tttatgtgta
     5461 tgattgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg ttgtgtatat
     5521 atatttatgg tagtgagagg caacgctccg gctcaggtgt caggttggtt tttgagacag
     5581 agtctttcgc ttagcttgga attaattcgt aatcatggtc atagctgttt cctgtgtgaa
     5641 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5701 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5761 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5821 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5881 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5941 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     6001 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     6061 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6121 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6181 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
     6241 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6301 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6361 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6421 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     6481 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6541 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6601 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6661 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6721 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6781 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6841 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6901 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6961 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7021 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7081 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7141 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7201 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7261 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7321 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7381 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7441 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7501 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7561 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7621 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7681 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7741 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     7801 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     7861 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     7921 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     7981 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac
     8041 cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca
     8101 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     8161 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     8221 aaacgacggc cagtgccaag ctagctttgc tcttaggagt ttcctaatac atcccaaact
     8281 caaatatata aagcatttga cttgttctat gccctagggg gcggggggaa gctaagccag
     8341 ctttttttaa catttaaaat gttaattcca ttttaaatgc acagatgttt ttatttcata
     8401 agggtttcaa tgtgcatgaa tgctgcaata ttcctgttac caaagctagt ataaataaaa
     8461 atagataaac gtggaaatta cttagagttt ctgtcattaa cgtttccttc ctcagttgac
     8521 aacataaatg cgctgctgag aagccagttt gcatctgtca ggatcaattt cccattatgc
     8581 cagtcatatt aattactagt caattagttg atttttattt ttgacatata catg
//
