LOCUS       sequence-430626-        8834 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 8834)
  AUTHORS   Yuno M, Nagata S, Fujita T, Fujii H
  TITLE     MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP
            analysis.
  JOURNAL   Biol Methods Protoc. 2021 Jul 9;6(1):bpab013. doi: 
            10.1093/biomethods/bpab013. eCollection 2021.
  PUBMED    34409168
REFERENCE   2  (bases 1 to 8834)
  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: "Biol 
            Methods Protoc."; date: "2021-07-9"; volume: "6(1):bpab013. doi"; 
            pages: " 10.1093/biomethods/bpab013. eCollection 2021"
COMMENT     Sequence Label: gRNA-hIRF-1 #12/pSIR-hCD2
FEATURES             Location/Qualifiers
     source          1..8834
                     /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        2120..2368
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2120..2140
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2291..2310
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2389..2464
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(2476..2500)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(5818..5834)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5818..5834)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5831..5853)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5842..5858
                     /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(5866..5872,5873..5890,5891..5896))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5911..5932
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6049..6066)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6220..6808)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6300..6319)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6979..7770,7771..7839))
                     /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     7602..7621
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7840..7944)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8012..8030
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8068..8090)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8190..8209
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8403..8425
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8417..8434
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8418..8434
                     /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 aagcttgtcg acggatccga
     2041 attcgccctt tgtacaaaaa agcaggcttt aaaggaacca attcagtcga ctggatccgg
     2101 taccaaggtc gggcaggaag agggcctatt tcccatgatt ccttcatatt tgcatatacg
     2161 atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa agatattagt
     2221 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     2281 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     2341 atatatcttg tggaaaggac gaaacaccgc gggggcgctg ggctgtccgt tttagagcta
     2401 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     2461 gtgctttttt tctagaccca gctttcttgt acaaagttgg cattaaaggg cgaattcgtc
     2521 gagggaagaa ataataaaga cattaaaatc acttttgtaa actttttaaa taagggaaat
     2581 tgattaaaac aacgcgaagt tgtaaaatgc catatactgc atacattttg tgggaaaaaa
     2641 tgtgcattta cctgctcacc aaattatctt attagctagc agtagagctc tttgtgtgat
     2701 gggaagatgg gataacagct gttgttactg tactggtttt tttttttaat atatttaaaa
     2761 aaaagagtgt ctcctaaagt gtaagcgcta tcatgctgga ctaatgtttt aatttaattt
     2821 aattgttgtc ttggtagaga tggggtctcg ctattttgct cacgcagatc tcaaactcct
     2881 gacgcccagg tacactccca tctcggcctc ctaaaatggt gggatcacag acgtcagcca
     2941 ccgagcccgg tcactttttt gtattcccca cagtattgat gtatatcttc tgcgttcaaa
     3001 agcaattttt taaagcctca taacgtggta acagaatact ttgcacatta caaaattcag
     3061 aacacggaaa caagaagctc gctttttttt cccccctatt tcggtttggc cctttagatt
     3121 tcccctcccc caccggggcg ggacttcccg ccgacttctt tcaggttctc agttcggtcc
     3181 gccaactgtc gtataaaggc gctgcctcag gccagaggcc tcacaaagcg cgtcgagaca
     3241 gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa gccctgcaaa
     3301 gtaaactgga tggctttctt gccgccaagg atctgatggc gcaggggatc aagatcgatc
     3361 tgatcaattc ctaagatgag ctttccatgt aaatttgtag ccagcttcct tctgattttc
     3421 aatgtttctt ccaaaggtgc agtctccaaa gagattacga atgccttgga aacctggggt
     3481 gccttgggtc aggacatcaa cttggacatt cctagttttc aaatgagtga tgatattgac
     3541 gatataaaat gggaaaaaac ttcagacaag aaaaagattg cacaattcag aaaagagaaa
     3601 gagactttca aggaaaaaga tacatataag ctatttaaaa atggaactct gaaaattaag
     3661 catctgaaga ccgatgatca ggatatctac aaggtatcaa tatatgatac aaaaggaaaa
     3721 aatgtgttgg aaaaaatatt tgatttgaag attcaagaga gggtctcaaa accaaagatc
     3781 tcctggactt gtatcaacac aaccctgacc tgtgaggtaa tgaatggaac tgaccccgaa
     3841 ttaaacctgt atcaagatgg gaaacatcta aaactttctc agagggtcat cacacacaag
     3901 tggaccacca gcctgagtgc aaaattcaag tgcacagcag ggaacaaagt cagcaaggaa
     3961 tccagtgtcg agcctgtcag ctgtccagag aaaggtctgg acatctatct catcattggc
     4021 atatgtggag gaggcagcct cttgatggtc tttgtggcac tgctcgtttt ctatatcacc
     4081 aaaaggaaaa aacagaggag tcggagaaat gatgaggagc tggagacaag agcccacaga
     4141 gtagctactg aagaaagggg ccggaagccc caacaaattc cagcttcaac ccctcagaat
     4201 ccagcaactt cccaacatcc tcctccacca cctggtcatc gttcccaggc acctagtcat
     4261 cgtcccccgc ctcctggaca ccgtgttcag caccagcctc agaagaggcc tcctgctccg
     4321 tcgggcacac aagttcacca gcagaaaggc ccgcccctcc ccagacctcg agttcagcca
     4381 aaacctcccc atggggcagc agaaaactca ttgtcccctt cctctaatta aaaaagatag
     4441 aaactgtctt tttcaataaa aagcactgtg gatttctgcc tctagggctg gatgatcctc
     4501 cagcgcgggg atctcatgct ggagatcccg cgattagtcc aatttgttaa agacaggata
     4561 tcagtggtcc aggctctagt tttgactcaa caatatcacc agctgaagcc tatagagtac
     4621 gagccataga taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac
     4681 cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaaat
     4741 acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac agggtcgacc
     4801 ggtcgaccgg tcgaccctag agaaccatca gatgtttcca gggtgcccca aggacctgaa
     4861 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc
     4921 ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc
     4981 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga
     5041 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg
     5101 gggtctttca catgcagcat gtatcaaaat taatttggtt ttttttctta agtatttaca
     5161 ttaaatggcc atagtactta aagttacatt ggcttccttg aaataaacat ggagtattca
     5221 gaatgtgtca taaatatttc taattttaag atagtatctc cattggcttt ctactttttc
     5281 ttttattttt ttttgtcctc tgtcttccat ttgttgttgt tgttgtttgt ttgtttgttt
     5341 gttggttggt tggttaattt ttttttaaag atcctacact atagttcaag ctagactatt
     5401 agctactctg taacccaggg tgaccttgaa gtcatgggta gcctgctgtt ttagccttcc
     5461 cacatctaag attacaggta tgagctatca tttttggtat attgattgat tgattgattg
     5521 atgtgtgtgt gtgtgattgt gtttgtgtgt gtgattgtgt atatgtgtgt atggttgtgt
     5581 gtgattgtgt gtatgtatgt ttgtgtgtga ttgtgtgtgt gtgattgtgc atgtgtgtgt
     5641 gtgtgattgt gtttatgtgt atgattgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt
     5701 gtgtgtgtgt gttgtgtata tatatttatg gtagtgagag gcaacgctcc ggctcaggtg
     5761 tcaggttggt ttttgagaca gagtctttca cttagcttgg aattaattcg taatcatgtc
     5821 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5881 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5941 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     6001 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     6061 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     6121 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     6181 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6241 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6301 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6361 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6421 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6481 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6541 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6601 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6661 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6721 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6781 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6841 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6901 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6961 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     7021 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     7081 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     7141 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     7201 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7261 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     7321 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7381 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7441 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7501 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7561 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     7621 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7681 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7741 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7801 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7861 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7921 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     7981 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     8041 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     8101 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     8161 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     8221 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca
     8281 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     8341 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     8401 ttcccagtca cgacgttgta aaacgacggc cagtgccaag ctagctttgc tcttaggagt
     8461 ttcctaatac atcccaaact caaatatata aagcatttga cttgttctat gccctagggg
     8521 gcggggggaa gctaagccag ctttttttaa catttaaaat gttaattcca ttttaaatgc
     8581 acagatgttt ttatttcata agggtttcaa tgtgcatgaa tgctgcaata ttcctgttac
     8641 caaagctagt ataaataaaa atagataaac gtggaaatta cttagagttt ctgtcattaa
     8701 cgtttccttc ctcagttgac aacataaatg cgctgctgag aagccagttt gcatctgtca
     8761 ggatcaattt cccattatgc cagtcatatt aattactagt caattagttg atttttattt
     8821 ttgacatata catg
//
