LOCUS       sequence-210556-        7974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR interference; works in conjunction with an sgRNA expression 
            vector to silence genes in the E. coli chromosome..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7974)
  TITLE     CRISPR interference and CRISPR single guide RNA
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7974)
  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: 
            "Unpublished"
COMMENT     Sequence Label: T1-araC-Pbad-D10A/H840A cas9 ABCD-pACYC-BB
FEATURES             Location/Qualifiers
     source          1..7974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(187..1065)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1092..1376
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1299..1318
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             join(1425..1451,1452..1454,1455..3941,3942..3944,
                     3945..5528)
                     /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"
     misc_feature    5541..5561
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      5606..5700
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      5742..6287
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     6188..6207
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6404..6421
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6813..6915
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             6916..7575
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(6982..7001)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(7796..7843)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     7839..7857
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(7895..7939)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     misc_feature    join(7959..7974,1..4)
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that start with ""ATG"""
ORIGIN
        1 ctagattttt gagataaaaa tgccagccga tcgggctggc attttgcctt taggatgtac
       61 acaatgagac agaagaggct agaactatag ctagcatgca taatgtgcct gtcaaatgga
      121 cgaagcaggg attctgcaaa ccctatgcta ctccgtcaag ccgtcaattg tctgattcgt
      181 taccaattat gacaacttga cggctacatc attcactttt tcttcacaac cggcacggaa
      241 ctcgctcggg ctggccccgg tgcatttttt aaatacccgc gagaaataga gttgatcgtc
      301 aaaaccaaca ttgcgaccga cggtggcgat aggcatccgg gtggtgctca aaagcagctt
      361 cgcctggctg atacgttggt cctcgcgcca gcttaagacg ctaatcccta actgctggcg
      421 gaaaagatgt gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgc tggcgatatc
      481 aaaattgctg tctgccaggt gatcgctgat gtactgacaa gcctcgcgta cccgattatc
      541 catcggtgga tggagcgact cgttaatcgc ttccatgcgc cgcagtaaca attgctcaag
      601 cagatttatc gccagcagct ccgaatagcg cccttcccct tgcccggcgt taatgatttg
      661 cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga accccgtatt
      721 ggcaaatatt gacggccagt taagccattc atgccagtag gcgcgcggac gaaagtaaac
      781 ccactggtga taccattcgc gagcctccgg atgacgaccg tagtgatgaa tctctcctgg
      841 cgggaacagc aaaatatcac ccggtcggca aacaaattct cgtccctgat ttttcaccac
      901 cccctgaccg cgaatggtga gattgagaat ataacctttc attcccagcg gtcggtcgat
      961 aaaaaaatcg agataaccgt tggcctcaat cggcgttaaa cccgccacca gatgggcatt
     1021 aaacgagtat cccggcagca ggggatcatt ttgcgcttca gccatacttt tcatactccc
     1081 gccattcaga gaagaaacca attgtccata ttgcatcaga cattgccgtc actgcgtctt
     1141 ttactggctc ttctcgctaa ccaaaccggt aaccccgctt attaaaagca ttctgtaaca
     1201 aagcgggacc aaagccatga caaaaacgcg taacaaaagt gtctataatc acggcagaaa
     1261 agtccacatt gattatttgc acggcgtcac actttgctat gccatagcat ttttatccat
     1321 aagattagcg gatcctacct gacgcttttt atcgcaactc tctactgttt ctccataccc
     1381 gttttttggg ctaacaggag gaattaacca tgggtgcggc ttcaatggac aaaaaatata
     1441 gcatcggcct cgcgattggt actaactccg tcggttgggc ggtgattact gatgaatata
     1501 aagtgccgag caagaaattt aaggtgctcg gcaacaccga ccgccacagc attaagaaaa
     1561 atctgattgg cgcgttgctt tttgattctg gtgaaacagc tgaagcgacg cgtctgaagc
     1621 gtaccgcgcg ccgtcgctat acccgccgca aaaaccgcat ttgctatttg caggagatat
     1681 ttagcaatga gatggcgaaa gtcgatgatt cgtttttcca tcgcctggaa gagagctttc
     1741 tggttgaaga agacaaaaaa cacgaacgcc atccgatttt tggcaatatt gtcgatgaag
     1801 ttgcttacca tgaaaagtac ccgaccattt atcacttacg caagaaatta gtagactcca
     1861 ccgataaagc tgatttacgt ctcatctatc tcgccctggc gcacatgatt aaattccgcg
     1921 gtcattttct cattgaaggc gacctcaatc ctgataacag tgatgtggac aagctgttca
     1981 tccagctcgt gcaaacctat aatcagctgt ttgaagaaaa cccgatcaat gccagcggcg
     2041 tggatgcaaa agcgatctta agcgcccgtt tgagtaaatc ccgccggctg gaaaatctta
     2101 ttgctcagtt accgggcgag aagaagaacg ggttgtttgg taacctgatt gcgctgtcac
     2161 tcggtctgac gccaaacttc aaaagtaatt tcgacctggc agaagatgct aaactgcaac
     2221 ttagcaaaga tacctacgat gacgacctgg ataatttatt ggcacagatc ggtgaccagt
     2281 atgcggatct cttcctggcg gcaaaaaatc tctcagatgc gattttactc tctgatattc
     2341 tgcgcgtgaa tacggaaatc acaaaagcgc cgttgagcgc aagcatgatc aaacgttatg
     2401 acgaacatca ccaggatttg actctgttaa aagcgctggt gcgtcagcaa cttccggaaa
     2461 aatacaaaga gattttcttc gatcagtcga aaaacggtta tgccggctat attgacggcg
     2521 gcgcttcgca ggaagagttt tacaagttta ttaaacccat tctggagaaa atggatggca
     2581 ccgaagagtt actggttaaa ctgaaccgtg aggacttgct gcgtaaacag cgcaccttcg
     2641 ataatggttc tatcccgcat cagattcatc tgggggaact gcacgccatc ctgcggcgtc
     2701 aggaagattt ctacccgttc cttaaagaca accgcgagaa aatcgaaaaa atcctcacgt
     2761 ttcgcattcc gtattatgtc ggcccgctgg cgcgtggtaa cagccgtttc gcctggatga
     2821 cccgaaaaag cgaagaaact attacgccgt ggaactttga agaagtggta gataagggtg
     2881 cctctgcgca aagttttatc gagcgcatga cgaactttga taagaatctg ccgaacgaaa
     2941 aggtactgcc aaaacactcg ctgctctatg agtatttcac cgtttataac gagttgacga
     3001 aagttaaata cgtcactgaa ggtatgcgta aacctgcctt tttaagtggc gaacaaaaaa
     3061 aggccattgt ggatctgctg ttcaaaacaa accggaaagt caccgtgaag caactgaaag
     3121 aggattactt caagaagatt gaatgcttcg acagcgtaga aatttccggc gttgaagatc
     3181 gctttaacgc cagtctgggc acttaccacg atctgctgaa gatcatcaag gataaggact
     3241 tcctggacaa tgaagagaat gaagatatac tggaagacat cgtgctgaca ttaacgctct
     3301 ttgaggatcg tgaaatgatt gaagagcgtc tgaaaactta tgcacattta tttgatgata
     3361 aagtaatgaa gcagctaaaa aggcgtcgtt acactggctg gggtcgacta tcgcgcaagt
     3421 tgatcaacgg cattcgtgat aaacagagcg gtaaaacgat tctcgatttt ttgaagagcg
     3481 acggttttgc caatcgcaat tttatgcagc ttatccatga tgacagctta acctttaaag
     3541 aagatattca gaaggcgcag gtgtccggtc agggcgatag cctgcatgag catatcgcga
     3601 acctggcggg ttcgccggca atcaaaaaag gtattcttca gacggtaaaa gtggttgacg
     3661 agctggtgaa agtgatgggc cgtcataaac cagaaaatat cgttattgaa atggcacggg
     3721 aaaaccagac cacccagaaa ggccagaaaa atagccgcga acgtatgaaa cgcatcgagg
     3781 aagggatcaa ggaacttggc agtcagatcc tgaaagagca cccggtggag aacacgcaat
     3841 tacagaacga aaaactctac ctttattacc tgcaaaacgg tcgcgatatg tatgttgatc
     3901 aagaattaga tattaatcgt ctttctgact acgacgttga tgcgattgta ccgcaatcct
     3961 tcctcaaaga cgattccatt gataacaaag tgctgactcg cagcgataaa aaccgtggca
     4021 agtccgataa cgtgccttct gaagaagtcg tgaaaaaaat gaaaaactac tggcgccagt
     4081 tgctgaatgc gaaactgatc actcagcgta aatttgacaa cctcaccaaa gccgaacgcg
     4141 gcgggctgag tgaactggat aaagcaggct ttattaaacg ccagctggtg gaaacgcgcc
     4201 aaattaccaa gcatgttgcc cagattctgg actcgcgaat gaataccaaa tacgatgaaa
     4261 acgataagct gatccgtgaa gtgaaagtga tcaccttgaa atcgaaactg gtcagcgatt
     4321 tccgtaaaga cttccagttc tataaggtac gtgaaattaa caactatcac catgcgcatg
     4381 acgcttatct taatgcagtg gtgggtacgg cgttaataaa aaaatatccg aagctcgaaa
     4441 gcgaatttgt ctacggcgat tataaagtct atgatgtccg taagatgatc gccaaatctg
     4501 agcaggaaat tgggaaagcc accgccaagt acttcttcta cagcaacatc atgaacttct
     4561 ttaaaaccga aatcacgcta gctaacggtg aaatccgcaa acgtccgtta atcgaaacca
     4621 acggcgaaac cggtgagatc gtctgggata aagggcgcga ctttgctacg gttcgtaagg
     4681 ttctgtcgat gccgcaggtt aacattgtta agaaaaccga ggtgcagacc ggcggtttca
     4741 gcaaagaaag tattttgccg aaacgtaact cggacaaatt gattgcccgc aaaaaggact
     4801 gggacccgaa aaaatatggc ggatttgaca gtccaaccgt cgcctacagc gtgctggtag
     4861 tggcgaaggt cgaaaaaggc aaatcaaaaa agctgaaaag cgtcaaagag ctgctgggta
     4921 tcaccatcat ggaacgtagc agcttcgaaa aaaatccaat cgactttctt gaagccaagg
     4981 gctacaaaga agtgaagaag gacttgatca tcaagctgcc aaagtattcc ctgtttgaac
     5041 tggagaacgg acgcaaacgg atgctggcat cggcgggcga attgcaaaaa ggtaatgaac
     5101 tggcactgcc gtctaaatat gtgaacttcc tgtatctggc cagccactat gaaaaactga
     5161 aaggcagccc ggaagataac gaacagaaac aactgtttgt tgagcagcac aaacattatc
     5221 tggatgagat cattgagcaa atcagcgagt tcagtaagcg cgttatcctt gccgatgcca
     5281 acctggataa agtgttgtct gcctataata aacatcgtga caaaccgatt cgcgaacagg
     5341 cggaaaacat catccacctg tttaccctga ccaatttggg cgcaccggcg gcgtttaaat
     5401 actttgacac cactatcgac cgtaaacgct acaccagcac caaagaagta cttgatgcca
     5461 cgctgattca tcaaagcata acgggcctgt acgaaacccg tattgatctg agccagttag
     5521 gtggtgataa gcttctcgag tactagtagc ggccgctgca gacatgtgag caaaaggcca
     5581 gcaaaaggcc aattagctga gcttggactc ctgttgatag atccagtaat gacctcagaa
     5641 ctccatctgg atttgttcag aacgctcggt tgccgccggg cgttttttat tggtgagaat
     5701 ccaagctaga aatattttat ctgattaata agatgatctt cttgagatcg ttttggtctg
     5761 cgcgtaatct cttgctctga aaacgaaaaa accgccttgc agggcggttt ttcgaaggtt
     5821 ctctgagcta ccaactcttt gaaccgaggt aactggcttg gaggagcgca gtcaccaaaa
     5881 cttgtccttt cagtttagcc ttaaccggcg catgacttca agactaactc ctctaaatca
     5941 attaccagtg gctgctgcca gtggtgcttt tgcatgtctt tccgggttgg actcaagacg
     6001 atagttaccg gataaggcgc agcggtcgga ctgaacgggg ggttcgtgca tacagtccag
     6061 cttggagcga actgcctacc cggaactgag tgtcaggcgt ggaatgagac aaacgcggcc
     6121 ataacagcgg aatgacaccg gtaaaccgaa aggcaggaac aggagagcgc acgagggagc
     6181 cgccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac cactgatttg
     6241 agcgtcagat ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaac ggctttgccg
     6301 cggccctctc acttccctgt taagtatctt cctggcatct tccaggaaat ctccgccccg
     6361 ttcgtaagcc atttccgctc gccgcagtcg aacgaccgag cgtagcgagt cagtgagcga
     6421 ggaagcggaa tatatcctgt atcacatatt ctgctgacgc accggtgcag ccttttttct
     6481 cctgccacat gaagcacttc actgacaccc tcatcagtgc caacatagta agccagtata
     6541 cactccgcta gcgctgatgt ccggcggtgc ttttgccgtt acgcaccacc ccgtcagtag
     6601 ctgaacagga gggacagctg atagaaacag aagccactgg agcacctcaa aaacaccatc
     6661 atacactaaa tcagtaagtt ggcagcatca cccgacgcac tttgcgccga ataaatacct
     6721 gtgacggaag atcacttcgc agaataaata aatcctggtg tccctgttga taccgggaag
     6781 ccctgggcca acttttggcg aaaatgagac gttgatcggc acgtaagagg ttccaacttt
     6841 caccataatg aaataagatc actaccgggc gtattttttg agttatcgag attttcagga
     6901 gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa
     6961 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag
     7021 accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt
     7081 tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga gttccgtatg
     7141 gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc
     7201 catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag
     7261 tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct
     7321 aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt
     7381 tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac tatgggcaaa
     7441 tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt
     7501 tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg
     7561 cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc tggttgctac
     7621 gcctgaataa gtgataataa gcggatgaat ggcagaaatt cgaaagcaaa ttcgacccgg
     7681 tcgtcggttc agggcagggt cgttaaatag ccgcttatgt ctattgctgg tttaccggtt
     7741 tattgactac cggaagcagt gtgaccgtgt gcttctcaaa tgcctgaggt ttcagcaaaa
     7801 aacccctcaa gacccgttta gaggccccaa ggggttatgc tagttattgc tcagcggtgg
     7861 cagcagccta ggttaattaa gctgcgctag tagacgagtc catgtgctgg cgttcaaatt
     7921 tcgcagcagc ggtttcttta ccagactcga cggagctcga attcgcggcc gctt
//
