LOCUS       sequence-322900-        7830 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  use as an auxiliary temperature-sensitive plasmid to successfully 
            transform pdCas9_CL plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7830)
  AUTHORS   Huang HH, Bellato M, Qian Y, Cardenas P, Pasotti L, Magni P, Del 
            Vecchio D
  TITLE     dCas9 regulator to neutralize competition in CRISPRi circuits.
  JOURNAL   Nat Commun. 2021 Mar 16;12(1):1692. doi: 10.1038/s41467-021-21772-6.
  PUBMED    33727557
REFERENCE   2  (bases 1 to 7830)
  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: "Nat 
            Commun."; date: "2021-03-16"; volume: "12(1):1692. doi"; pages: " 
            10.1038/s41467-021-21772-6"
COMMENT     Sequence Label: pAUX_OL
FEATURES             Location/Qualifiers
     source          1..7830
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        74..149
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    204..225
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        226..260
                     /label=BBa_J23116
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.16"
     RBS             269..280
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(287..313,314..316,317..2803,2804..2806,2807..4393)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     terminator      4417..4488
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4504..4531
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      4549..4793
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(4916..5866)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      5914..6136
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(6758..7549,7550..7618))
                     /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     7381..7400
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7619..7723)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7810..7828
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ggcgcgccaa aaagagtatt gacttcgcat ctttttgtac ctataataga ttcagtattg
       61 actattaatc atcgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
      121 aacttgaaaa agtggcaccg agtcggtgct ttttttccaa ttattgaaca ccctaacggg
      181 tgtttttttt tttttggtct cccgaattcg cggccgcttc tagagttgac agctagctca
      241 gtcctaggga ctatgctagc tactagagaa agaggagaaa ggatctatgg ataagaaata
      301 ctcaataggc ttagctatcg gcacaaatag cgtcggatgg gcggtgatca ctgatgaata
      361 taaggttccg tctaaaaagt tcaaggttct gggaaataca gaccgccaca gtatcaaaaa
      421 aaatcttata ggggctcttt tatttgacag tggagagaca gcggaagcga ctcgtctcaa
      481 acggacagct cgtagaaggt atacacgtcg gaagaatcgt atttgttatc tacaggagat
      541 tttttcaaat gagatggcga aagtagatga tagtttcttt catcgacttg aagagtcttt
      601 tttggtggaa gaagacaaga agcatgaacg tcatcctatt tttggaaata tagtagatga
      661 agttgcttat catgagaaat atccaactat ctatcatctg cgaaaaaaat tggtagattc
      721 tactgataaa gcggatttgc gcttaatcta tttggcctta gcgcatatga ttaagtttcg
      781 tggtcatttt ttgattgagg gagatttaaa tcctgataat agtgatgtgg acaaactatt
      841 tatccagttg gtacaaacct acaatcaatt atttgaagaa aaccctatta acgcaagtgg
      901 agtagatgct aaagcgattc tttctgcacg attgagtaaa tcaagacgat tagaaaatct
      961 cattgctcag ctccccggtg agaagaaaaa tggcttattt gggaatctca ttgctttgtc
     1021 attgggtttg acccctaatt ttaaatcaaa ttttgatttg gcagaagatg ctaaattaca
     1081 gctttcaaaa gatacttacg atgatgattt agataattta ttggcgcaaa ttggagatca
     1141 atatgctgat ttgtttttgg cagctaagaa tttatcagat gctattttac tttcagatat
     1201 cctaagagta aatactgaaa taactaaggc tcccctatca gcttcaatga ttaaacgcta
     1261 cgatgaacat catcaagact tgactctttt aaaagcttta gttcgacaac aacttccaga
     1321 aaagtataaa gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt atattgatgg
     1381 gggagctagc caagaagaat tttataaatt tatcaaacca attttagaaa aaatggatgg
     1441 tactgaggaa ttattggtga aactaaatcg tgaagatttg ctgcgcaagc aacggacctt
     1501 tgacaacggc tctattcccc atcaaattca cttgggtgag ctgcatgcta ttttgagaag
     1561 acaagaagac ttttatccat ttttaaaaga caatcgtgag aagattgaaa aaatcttgac
     1621 ttttcgaatt ccttattatg ttggtccatt ggcgcgtggc aatagtcgtt ttgcatggat
     1681 gactcggaag tctgaagaaa caattacccc atggaatttt gaagaagttg tcgataaagg
     1741 tgcttcagct caatcattta ttgaacgcat gacaaacttt gataaaaatc ttccaaatga
     1801 aaaagtacta ccaaaacata gtttgcttta tgagtatttt acggtttata acgaattgac
     1861 aaaggtcaaa tatgttactg aaggaatgcg aaaaccagca tttctttcag gtgaacagaa
     1921 gaaagccatt gttgatttac tcttcaaaac aaatcgaaaa gtaaccgtta agcaattaaa
     1981 agaagattat ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag gagttgaaga
     2041 tagatttaat gcttcattag gtacctacca tgatttgcta aaaattatta aagataaaga
     2101 ttttttggat aatgaagaaa atgaagatat cttagaggat attgttttaa cattgacctt
     2161 atttgaagat agggagatga ttgaggaaag acttaaaaca tatgctcacc tctttgatga
     2221 taaggtgatg aaacagctta aacgtcgccg ttatactggt tggggacgtt tgtctcgaaa
     2281 attgattaat ggtattaggg ataagcaatc tggcaaaaca atattagatt ttttgaaatc
     2341 agatggtttt gccaatcgca attttatgca gctgatccat gatgatagtt tgacatttaa
     2401 agaagacatt caaaaagcac aagtgtctgg acaaggcgat agtttacatg aacatattgc
     2461 aaatttagct ggtagccctg ctattaaaaa aggtatttta cagactgtaa aagttgttga
     2521 tgaattggtc aaagtaatgg ggcggcataa gccagaaaat atcgttattg aaatggcacg
     2581 tgaaaatcag acaactcaaa agggccagaa aaattcgcga gagcgtatga aacgaatcga
     2641 agaaggtatc aaagaattag gaagtcagat tcttaaagag catcctgttg aaaatactca
     2701 attgcaaaat gaaaagctct atctctatta tctccaaaat ggaagagaca tgtatgtgga
     2761 ccaagaatta gatattaatc gtttaagtga ttatgatgtc gatgccattg ttccacaaag
     2821 tttccttaaa gacgattcaa tagacaataa ggtcttaacg cgttctgata aaaatcgtgg
     2881 taaatcggat aacgttccaa gtgaagaagt agtcaaaaag atgaaaaact attggagaca
     2941 acttctaaac gccaagttaa tcactcaacg taagtttgat aatttaacga aagctgaacg
     3001 tggaggtttg agtgaacttg ataaagctgg ttttatcaaa cgccaattgg ttgaaactcg
     3061 ccaaatcact aagcatgtgg cacaaatttt ggatagtcgc atgaatacta aatacgatga
     3121 aaatgataaa cttattcgag aggttaaagt gattacctta aaatctaaat tagtttctga
     3181 cttccgaaaa gatttccaat tctataaagt acgtgagatt aacaattacc atcatgccca
     3241 tgatgcgtat ctaaatgccg tcgttggaac tgctttgatt aagaaatatc caaaacttga
     3301 atcggagttt gtctatggtg attataaagt ttatgatgtt cgtaaaatga ttgctaagtc
     3361 tgagcaagaa ataggcaaag caaccgcaaa atatttcttt tactctaata tcatgaactt
     3421 cttcaaaaca gaaattacac ttgcaaatgg agagattcgc aaacgccctc taatcgaaac
     3481 taatggggaa actggagaaa ttgtctggga taaagggcga gattttgcca cagtgcgcaa
     3541 agtattgtcc atgccccaag tcaatattgt caagaaaaca gaagtacaga caggcggatt
     3601 ctccaaggag tcaattttac caaaaagaaa ttcggacaag cttattgctc gtaaaaaaga
     3661 ctgggatcca aaaaaatatg gtggttttga tagtccaacg gtagcttatt cagtcctagt
     3721 ggttgctaag gtggaaaaag ggaaatcgaa gaagttaaaa tccgttaaag agttactagg
     3781 gatcacaatt atggaaagaa gttcctttga aaaaaatccg attgactttt tagaagctaa
     3841 aggatataag gaagttaaaa aagacttaat cattaaacta cctaaatata gtctttttga
     3901 gttagaaaac ggtcgtaaac ggatgctggc tagtgccgga gaattacaaa aaggaaatga
     3961 gctggctctg ccaagcaaat atgtgaattt tttatattta gctagtcatt atgaaaagtt
     4021 gaagggtagt ccagaagata acgaacaaaa acaattgttt gtggagcagc ataagcatta
     4081 tttagatgag attattgagc aaatcagtga attttctaag cgtgttattt tagcagatgc
     4141 caatttagat aaagttctta gtgcatataa caaacataga gacaaaccaa tacgtgaaca
     4201 agcagaaaat attattcatt tatttacgtt gacgaatctt ggagctcccg ctgcttttaa
     4261 atattttgat acaacaattg atcgtaaacg atatacgtct acaaaagaag ttttagatgc
     4321 cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt tgagtcagct
     4381 aggaggtgac taactcgagt aaggatctcc aggcatcaaa taaaacgaaa ggctcagtcg
     4441 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac
     4501 actggctcac cttcgggtgg gcctttctgc gtttataaat ggcgatgacg catcctcacg
     4561 ataatatccg ggtaggcgca atcactttcg tctactccgt tacaaagcga ggctgggtat
     4621 ttcccggcct ttctgttatc cgaaatccac tgaaagcaca gcggctggct gaggagataa
     4681 ataataaacg aggggctgta tgcacaaagc atcttctgtt gagttaagaa cgagtatcga
     4741 gatggcacat agccttgctc aaattggaat caggtttgtg ccaataccag tagaaacaga
     4801 cgaagaatcc atgggtatgg acagttttcc ctttgatatg taacggtgaa cagttgttct
     4861 acttttgttt gttagtcttg atgcttcact gatagataca agagccataa gaacctcaga
     4921 tccttccgta tttagccagt atgttctcta gtgtggttcg ttgtttttgc gtgagccatg
     4981 agaacgaacc attgagatca tacttacttt gcatgtcact caaaaatttt gcctcaaaac
     5041 tggtgagctg aatttttgca gttaaagcat cgtgtagtgt ttttcttagt ccgttatgta
     5101 ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc attcattttt atctggttgt
     5161 tctcaagttc ggttacgaga tccatttgtc tatctagttc aacttggaaa atcaacgtat
     5221 cagtcgggcg gcctcgctta tcaaccacca atttcatatt gctgtaagtg tttaaatctt
     5281 tacttattgg tttcaaaacc cattggttaa gccttttaaa ctcatggtag ttattttcaa
     5341 gcattaacat gaacttaaat tcatcaaggc taatctctat atttgccttg tgagttttct
     5401 tttgtgttag ttcttttaat aaccactcat aaatcctcat agagtatttg ttttcaaaag
     5461 acttaacatg ttccagatta tattttatga atttttttaa ctggaaaaga taaggcaata
     5521 tctcttcact aaaaactaat tctaattttt cgcttgagaa cttggcatag tttgtccact
     5581 ggaaaatctc aaagccttta accaaaggat tcctgatttc cacagttctc gtcatcagct
     5641 ctctggttgc tttagctaat acaccataag cattttccct actgatgttc atcatctgaa
     5701 cgtattggtt ataagtgaac gataccgtcc gttctttcct tgtagggttt tcaatcgtgg
     5761 ggttgagtag tgccacacag cataaaatta gcttggtttc atgctccgtt aagtcatagc
     5821 gactaatcgc tagttcattt gctttgaaaa caactaattc agacatacat ctcaattggt
     5881 ctaggtgatt ttaatcacta taccaattga gatgggctag tcaatgataa ttactagtcc
     5941 ttttcctttg agttgtgggt atctgtaaat tctgctagac ctttgctgga aaacttgtaa
     6001 attctgctag accctctgta aattccgcta gacctttgtg tgtttttttt gtttatattc
     6061 aagtggttat aatttataga ataaagaaag aataaaaaaa gataaaaaga atagatccca
     6121 gccctgtgta taactcacta ctttagtcag ttccgcagta ttacaaaagg atgtcgcaaa
     6181 cgctgtttgc tcctctacaa aacagacctt aaaaccctaa aggcttaagt agcaccctcg
     6241 caagctcggt tgcggccgca atcgggcaaa tcgctgaata ttccttttgt ctccgaccat
     6301 caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca cggctctggc
     6361 agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa ggaaactacc
     6421 cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg gtctgctatg
     6481 tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc cagtctgacc
     6541 acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt aaggcagcgg
     6601 tatcatcaac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6661 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6721 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6781 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6841 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6901 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6961 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     7021 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     7081 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     7141 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7201 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7261 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7321 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7381 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7441 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7501 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7561 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7621 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7681 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7741 tgccacctgc atcgatttat gccacctgac gtctaagaaa ccattattat catgacatta
     7801 acctataaaa ataggcgtat cacgaggcag
//
