LOCUS       sequence-446712-        8834 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini-Tn7 dCas9 expression vector - AraC/pBAD promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8834)
  AUTHORS   Roghair Stroud MN, Vang DX, Halverson LJ
  TITLE     Optimized CRISPR Interference System for Investigating Pseudomonas 
            alloputida Genes Involved in Rhizosphere Microbiome Assembly.
  JOURNAL   ACS Synth Biol. 2024 Aug 20. doi: 10.1021/acssynbio.4c00312.
  PUBMED    39163848
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: "ACS Synth 
            Biol. 2024 Aug 20. doi: 10.1021/acssynbio.4c00312."
COMMENT     Sequence Label: pMRS-AraC-dCas9
FEATURES             Location/Qualifiers
     source          1..8834
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(51..929)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        956..1240
                     /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     1163..1182
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1319..1327
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     terminator      5489..5560
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(5563..5579)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5563..5579)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      5622..5716
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(6018..6037)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6029..6844
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     oriT            7011..7119
                     /note="incP origin of transfer"
     rep_origin      7141..7529
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     mobile_element  7538..7703
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     terminator      complement(7717..7803)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    7838..7885
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(8015..8548)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(8718..8723,8724..8740,8741..8746))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    8787..8834
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
        1 agagcgcttt tgaagctaat tcgatcatgc atgagcgacg tcttaattaa ttatgacaac
       61 ttgacggcta catcattcac tttttcttca caaccggcac ggaactcgct cgggctggcc
      121 ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc aacattgcga
      181 ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg gctgatacgt
      241 tggtcctcgc gccagcttaa gacgctaatc cctaactgct ggcggaaaag atgtgacaga
      301 cgcgacggcg acaagcaaac atgctgtgcg acgctggcga tatcaaaatt gctgtctgcc
      361 aggtgatcgc tgatgtactg acaagcctcg cgtacccgat tatccatcgg tggatggagc
      421 gactcgttaa tcgcttccat gcgccgcagt aacaattgct caagcagatt tatcgccagc
      481 agctccgaat agcgcccttc cccttgcccg gcgttaatga tttgcccaaa caggtcgctg
      541 aaatgcggct ggtgcgcttc atccgggcga aagaaccccg tattggcaaa tattgacggc
      601 cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt aaacccactg gtgataccat
      661 tcgcgagcct ccggatgacg accgtagtga tgaatctctc ctggcgggaa cagcaaaata
      721 tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca ccaccccctg accgcgaatg
      781 gtgagattga gaatataacc tttcattccc agcggtcggt cgataaaaaa atcgagataa
      841 ccgttggcct caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc
      901 agcaggggat cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa
      961 accaattgtc catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg
     1021 ctaaccaaac cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc
     1081 atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat
     1141 ttgcacggcg tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct
     1201 acctgacgct ttttatcgca actctctact gtttctccat accccctagg gcccaagttc
     1261 acttaaaaag gagatcaaca atgaaagcaa ttttcgtact gaaacatctt aatcatgcta
     1321 aggaggtacc gctagcggtt atagaaggag atatacatat ggataaaaag tacagcatcg
     1381 ggctcgcgat cgggaccaac tccgtggggt gggccgtcat cacggacgag tataaggtcc
     1441 cgtcgaagaa attcaaggtc ctcgggaaca cggatcggca tagcatcaaa aaagacctga
     1501 tcggggccct cctgttcgac tccggcgaga ccgcggaggc gacgcgcctc aagcgcacgg
     1561 cccggcgccg ctatacgcgg cgcaaaaatc ggatctgcta tctccaagag atcttcagca
     1621 atgagatggc gaaagtggat gactccttct tccaccggct cgaagagtcg ttcctggtcg
     1681 aggaagataa aaagcatgag agccatccga tcttcgggaa tatcgtggac gaggtggcct
     1741 accatgagaa atacccgacc atctatcatc tgcggaagaa actggtggac tccaccgaca
     1801 aggcggacct ccgcctgatc tatctcgcgc tggcccatat gatcaagttc cgcgggcact
     1861 tcctgatcga gggggatctc aaccccgata actcggatgt ggacaaactg ttcatccaac
     1921 tcgtccagac ctataatcaa ctgttcgaag agaatccgat caatgccagc ggcgtcgatg
     1981 ccaaggcgat cctctcggcc cggctctcca agtcgcggcg gctcgaaaat ctgatcgccc
     2041 aactccccgg cgagaagaag aatggcctgt tcgggaacct catcgccctc tccctggggc
     2101 tgacccccaa tttcaaaagc aacttcgacc tggccgaaga tgcgaaactc cagctgtcca
     2161 aggataccta cgacgacgat ctggacaacc tcctggcgca aatcggggat cagtatgccg
     2221 atctcttcct ggcggccaaa aatctctccg atgcgatcct cctgtcggat atcctgcggg
     2281 tgaataccga gatcaccaaa gcccccctga gcgcctcgat gatcaaacgc tacgacgagc
     2341 atcatcaaga cctgaccctc ctgaaggccc tcgtgcgcca gcagctgccg gagaaatata
     2401 aagaaatctt cttcgatcag tccaaaaatg ggtatgcggg gtacatcgac ggcggggcct
     2461 cgcaggaaga gttctataag ttcatcaaac cgatcctgga aaaaatggat gggaccgaag
     2521 agctcctggt gaaactcaac cgcgaagacc tcctgcggaa acagcgcacg ttcgacaacg
     2581 gctccatccc ccatcaaatc cacctcgggg agctccatgc gatcctccgc cggcaggaag
     2641 atttctaccc cttcctcaaa gataaccggg aaaaaatcga gaaaatcctc accttccgga
     2701 tcccctacta cgtcggcccg ctggcgcggg ggaactcccg gttcgcgtgg atgacgcgca
     2761 agtcggagga gacgatcacc ccctggaatt tcgaagaggt ggtggataag ggcgcctcgg
     2821 cgcaaagctt catcgaacgg atgacgaatt tcgacaaaaa cctcccgaat gagaaagtcc
     2881 tgcccaagca ttcgctgctc tacgaatatt tcaccgtgta caatgaactg acgaaagtca
     2941 agtatgtcac cgagggcatg cgcaaacccg ccttcctgtc gggggaacag aaaaaggcga
     3001 tcgtggatct gctgttcaaa accaatcgca aggtcacggt gaagcagctc aaagaagact
     3061 atttcaaaaa aatcgagtgc ttcgattccg tggagatctc cggcgtggaa gatcggttca
     3121 acgcgtcgct ggggacctat catgatctgc tgaaaatcat caaagataag gacttcctgg
     3181 acaacgaaga aaatgaagac atcctggagg acatcgtcct cacgctgacg ctgttcgagg
     3241 atcgcgagat gatcgaagag cgactgaaga cgtatgccca tctcttcgac gataaagtga
     3301 tgaaacaact caaacgccgg cggtacacgg gctgggggcg cctgtcccgc aagctgatca
     3361 acgggatccg ggacaaacaa agtgggaaaa cgatcctcga cttcctcaag tcggatggct
     3421 tcgcgaaccg gaacttcatg caactcatcc atgacgacag cctcaccttc aaggaagata
     3481 tccaaaaagc ccaagtctcc ggccagggcg actccctgca cgaacatatc gcgaacctgg
     3541 cgggctcgcc cgccatcaag aaaggcatcc tccagacggt caaagtggtc gacgaactgg
     3601 tgaaagtgat ggggcggcat aaacccgaga atatcgtcat cgaaatggcc cgcgagaacc
     3661 aaaccacgca gaaaggccag aaaaatagcc gggagcgcat gaagcgcatc gaggaaggca
     3721 tcaaggagct gggcagccag atcctcaagg agcatccggt cgaaaacacc caactccaaa
     3781 atgagaagct gtatctctac tatctgcaaa atgggcgcga tatgtacgtc gatcaggagc
     3841 tcgatatcaa ccgcctctcc gactacgatg tggatgcgat cgtcccccag tccttcctga
     3901 aagatgacag catcgataac aaggtgctca cccggtcgga caaaaatcgc gggaagtccg
     3961 ataatgtccc gtccgaagaa gtggtgaaaa agatgaagaa ctactggcgg caactcctca
     4021 acgcgaagct gatcacccag cgcaaattcg ataacctcac caaggccgag cggggcgggc
     4081 tctcggagct cgataaggcc ggcttcatca agcgccagct ggtcgagacg cgccaaatca
     4141 cgaagcacgt ggcgcaaatc ctcgatagcc ggatgaatac caagtatgac gagaatgata
     4201 aactcatccg ggaagtgaaa gtgatcacgc tgaaaagcaa actcgtgtcc gacttccgga
     4261 aggatttcca attctataaa gtgcgcgaaa tcaacaacta tcatcacgcg catgacgcgt
     4321 acctcaatgc cgtcgtgggc acggccctca tcaagaagta cccgaagctg gaaagcgaat
     4381 tcgtctacgg cgattacaaa gtgtacgatg tgcgcaaaat gatcgcgaaa tcggaacaag
     4441 aaatcggcaa ggcgaccgcg aagtacttct tctattcgaa tatcatgaat ttcttcaaaa
     4501 ccgaaatcac cctcgcgaac ggcgaaatcc gcaaacgccc gctcatcgag acgaacgggg
     4561 agaccgggga aatcgtgtgg gacaaggggc gcgacttcgc caccgtgcgg aaagtcctgt
     4621 ccatgccgca agtgaatatc gtgaaaaaaa ccgaagtgca aaccggcggc ttctcgaagg
     4681 aaagcatcct ccccaaacgc aacagcgata aactgatcgc ccggaagaag gactgggacc
     4741 ccaaaaaata tgggggcttc gactcgccga ccgtggcgta ctcggtcctc gtggtggcga
     4801 aggtcgaaaa agggaaaagc aaaaaactga aaagcgtcaa ggaactgctc ggcatcacga
     4861 tcatggagcg ctcctccttc gaaaaaaacc cgatcgattt cctggaggcg aaaggctaca
     4921 aggaggtcaa aaaggacctg atcatcaagc tccccaaata tagcctgttc gaactggaaa
     4981 atggccgcaa gcggatgctg gcctccgcgg gcgagctgca aaaggggaac gaactcgcgc
     5041 tcccgagcaa atacgtcaac ttcctctatc tcgcgtcgca ttatgaaaag ctgaagggga
     5101 gccccgaata taatgaacaa aaacagctgt tcgtggaaca acataaacac tatctcgatg
     5161 agatcatcga gcaaatctcg gagttctcca aacgggtcat cctcgcggac gccaatctcg
     5221 ataaggtgct gtcggcgtac aataaacatc gcgataagcc gatccgcgag caagccgaga
     5281 acatcatcca cctcttcacg ctgacgaatc tgggcgcgcc ggccgccttc aagtacttcg
     5341 acacgacgat cgatcggaag cggtacacct cgacgaaaga agtgctcgac gccacgctga
     5401 tccaccaatc catcacgggt ctgtatgaaa cccgcatcga cctctcgcaa ctcggcgggg
     5461 actaactcga gtaaggatct ccaggcatca aataaaacga aaggctcagt cgaaagactg
     5521 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc gggatccact agttctagag
     5581 cggccgcgtc gtgactggga aaaccctggc gactagtctt ggactcctgt tgatagatcc
     5641 agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt
     5701 ttttattggt gagaatccag ccacgcccct ctttaatacg acgggcaatt tgcacttcag
     5761 aaaatgaaga gtttgcttta gccataacaa aagtccagta tgctttttca cagcataact
     5821 ggactgattt cagtttacaa ctattctgtc tagtttaaga ctttattgtc atagtttaga
     5881 tctattttgt tcagtttaag actttattgt ccgcccacaa tttaaatttg tgtctcaaaa
     5941 tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc
     6001 ttacataaac agtaatacaa ggggtgttat gagccatatt cagcgtgaaa cgagctgtag
     6061 ccgtccgcgt ctgaacagca acatggatgc ggatctgtat ggctataaat gggcgcgtga
     6121 taacgtgggt cagagcggcg cgaccattta tcgtctgtat ggcaaaccgg atgcgccgga
     6181 actgtttctg aaacatggca aaggcagcgt ggcgaacgat gtgaccgatg aaatggtgcg
     6241 tctgaactgg ctgaccgaat ttatgccgct gccgaccatt aaacatttta ttcgcacccc
     6301 ggatgatgcg tggctgctga ccaccgcgat tccgggcaaa accgcgtttc aggtgctgga
     6361 agaatatccg gatagcggcg aaaacattgt ggatgcgctg gccgtgtttc tgcgtcgtct
     6421 gcatagcatt ccggtgtgca actgcccgtt taacagcgat cgtgtgtttc gtctggccca
     6481 ggcgcagagc cgtatgaaca acggcctggt ggatgcgagc gattttgatg atgaacgtaa
     6541 cggctggccg gtggaacagg tgtggaaaga aatgcataaa ctgctgccgt ttagcccgga
     6601 tagcgtggtg acccacggcg attttagcct ggataacctg attttcgatg aaggcaaact
     6661 gattggctgc attgatgtgg gccgtgtggg cattgcggat cgttatcagg atctggccat
     6721 tctgtggaac tgcctgggcg aatttagccc gagcctgcaa aaacgtctgt ttcagaaata
     6781 tggcattgat aatccggata tgaacaaact gcaatttcat ctgatgctgg atgaattttt
     6841 ctaataatta attggaccgc ggtccgcgcg ttgtcctttt ccgctgcata accctgcttc
     6901 ggggtcatta tagcgatttt ttcggtatat ccatcctttt tcgcacgata tacaggattt
     6961 tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc gggcaggata
     7021 ggtgaagtag gcccacccgc gagcgggtgt tccttcttca ctgtccctta ttcgcacctg
     7081 gcggtgctca acgggaatcc tgctctgcga ggctggccgt aggccggccg gcgcgcccca
     7141 tgtcagccgt taagtgttcc tgtgtcactc aaaattgctt tgagaggctc taagggcttc
     7201 tcagtgcgtt acatccctgg cttgttgtcc acaaccgtta aaccttaaag gctttaaaag
     7261 ccttatatat tctttttttt cttataaaac ttaaaacctt agaggctatt taagttgctg
     7321 atttatatta attttattgt tcaaacatga gagcttagta cgtgaaacat gagagcttag
     7381 tacgttagcc atgagagctt agtacgttag ccatgagggt ttagttcgtt aaacatgaga
     7441 gcttagtacg ttaaacatga gagcttagta cgtgaaacat gagagcttag tacgtactat
     7501 caacaggttg aactgctgat cttcagatcg gcgcgcctgt gggcggacaa aatagttggg
     7561 aactgggagg ggtggaaatg gagtttttaa ggattattta gggaagagtg acaaaataga
     7621 tgggaactgg gtgtagcgtc gtaagctaat acgaaaatta aaaatgacaa aatagtttgg
     7681 aactagattt cacttatctg gtttctaggg cggcggattt gtcctactca ggagagcgtt
     7741 caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat
     7801 ttgatgccga cgtccggacg atcgaattgg ggatcttgaa gtacctattc cgaagttcct
     7861 attctctaga aagtatagga acttcagagc gcttttgaag ctgatgtgct taaaaactta
     7921 ctcaatggaa ttctagatcg aattggccgc ggcgttgtga caatttaccg aacaactccg
     7981 cggccgggaa gccgatctcg gcttgaacga attgttaggt ggcggtactt gggtcgatat
     8041 caaagtgcat cacttcttcc cgtatgccca actttgtata gagagccact gcgggatcgt
     8101 caccgtaatc tgcttgcacg tagatcacat aagcaccaag cgcgttggcc tcatgcttga
     8161 ggagattgat gagcgcggtg gcaatgccct gcctccggtg ctcgccggag actgcgagat
     8221 catagatata gatctcacta cgcggctgct caaacttggg cagaacgtaa gccgcgagag
     8281 cgccaacaac cgcttcttgg tcgaaggcag caagcgcgat gaatgtctta ctacggagca
     8341 agttcccgag gtaatcggag tccggctgat gttgggagta ggtggctacg tctccgaact
     8401 cacgaccgaa aagatcaaga gcagcccgca tggatttgac ttggtcaggg ccgagcctac
     8461 atgtgcgaat gatgcccata cttgagccac ctaactttgt tttagggcga ctgccctgct
     8521 gcgtaacatc gttgctgctc cataacatca aacatcgacc cacggcgtaa cgcgcttgct
     8581 gcttggatgc ccgaggcata gactgtacaa aaaaacagtc ataacaagcc atgaaaaccg
     8641 ccactgcgcc gttaccaccg ctgcgttcgg tcaaggttct ggaccagttg cgtgagcgca
     8701 tacgctactt gcattacagt ttacgaaccg aacaggctta tgtcaatccg atccattgct
     8761 gttgacaaag ggaatcaggg gatcttgaag ttcctattcc gaagttccta ttctctagaa
     8821 agtataggaa cttc
//
