LOCUS       sequence-440945-        7688 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini-Tn7 dCas9 expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7688)
  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 7688)
  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-dCas9
FEATURES             Location/Qualifiers
     source          1..7688
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..73
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     RBS             154..162
                     /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      4324..4395
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(4398..4414)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4398..4414)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      4457..4551
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(4853..4872)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4864..5679
                     /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            5846..5954
                     /note="incP origin of transfer"
     rep_origin      5976..6364
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     mobile_element  6373..6538
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     terminator      complement(6552..6638)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    6673..6720
                     /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(6850..7383)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(7572..7577,7578..7594,7595..7600))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    7641..7688
                     /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 agcggataac
       61 aatttcacac aggaggccgc ctagggccca agttcactta aaaaggagat caacaatgaa
      121 agcaattttc gtactgaaac atcttaatca tgctaaggag gtaccgctag cggttataga
      181 aggagatata catatggata aaaagtacag catcgggctc gcgatcggga ccaactccgt
      241 ggggtgggcc gtcatcacgg acgagtataa ggtcccgtcg aagaaattca aggtcctcgg
      301 gaacacggat cggcatagca tcaaaaaaga cctgatcggg gccctcctgt tcgactccgg
      361 cgagaccgcg gaggcgacgc gcctcaagcg cacggcccgg cgccgctata cgcggcgcaa
      421 aaatcggatc tgctatctcc aagagatctt cagcaatgag atggcgaaag tggatgactc
      481 cttcttccac cggctcgaag agtcgttcct ggtcgaggaa gataaaaagc atgagagcca
      541 tccgatcttc gggaatatcg tggacgaggt ggcctaccat gagaaatacc cgaccatcta
      601 tcatctgcgg aagaaactgg tggactccac cgacaaggcg gacctccgcc tgatctatct
      661 cgcgctggcc catatgatca agttccgcgg gcacttcctg atcgaggggg atctcaaccc
      721 cgataactcg gatgtggaca aactgttcat ccaactcgtc cagacctata atcaactgtt
      781 cgaagagaat ccgatcaatg ccagcggcgt cgatgccaag gcgatcctct cggcccggct
      841 ctccaagtcg cggcggctcg aaaatctgat cgcccaactc cccggcgaga agaagaatgg
      901 cctgttcggg aacctcatcg ccctctccct ggggctgacc cccaatttca aaagcaactt
      961 cgacctggcc gaagatgcga aactccagct gtccaaggat acctacgacg acgatctgga
     1021 caacctcctg gcgcaaatcg gggatcagta tgccgatctc ttcctggcgg ccaaaaatct
     1081 ctccgatgcg atcctcctgt cggatatcct gcgggtgaat accgagatca ccaaagcccc
     1141 cctgagcgcc tcgatgatca aacgctacga cgagcatcat caagacctga ccctcctgaa
     1201 ggccctcgtg cgccagcagc tgccggagaa atataaagaa atcttcttcg atcagtccaa
     1261 aaatgggtat gcggggtaca tcgacggcgg ggcctcgcag gaagagttct ataagttcat
     1321 caaaccgatc ctggaaaaaa tggatgggac cgaagagctc ctggtgaaac tcaaccgcga
     1381 agacctcctg cggaaacagc gcacgttcga caacggctcc atcccccatc aaatccacct
     1441 cggggagctc catgcgatcc tccgccggca ggaagatttc taccccttcc tcaaagataa
     1501 ccgggaaaaa atcgagaaaa tcctcacctt ccggatcccc tactacgtcg gcccgctggc
     1561 gcgggggaac tcccggttcg cgtggatgac gcgcaagtcg gaggagacga tcaccccctg
     1621 gaatttcgaa gaggtggtgg ataagggcgc ctcggcgcaa agcttcatcg aacggatgac
     1681 gaatttcgac aaaaacctcc cgaatgagaa agtcctgccc aagcattcgc tgctctacga
     1741 atatttcacc gtgtacaatg aactgacgaa agtcaagtat gtcaccgagg gcatgcgcaa
     1801 acccgccttc ctgtcggggg aacagaaaaa ggcgatcgtg gatctgctgt tcaaaaccaa
     1861 tcgcaaggtc acggtgaagc agctcaaaga agactatttc aaaaaaatcg agtgcttcga
     1921 ttccgtggag atctccggcg tggaagatcg gttcaacgcg tcgctgggga cctatcatga
     1981 tctgctgaaa atcatcaaag ataaggactt cctggacaac gaagaaaatg aagacatcct
     2041 ggaggacatc gtcctcacgc tgacgctgtt cgaggatcgc gagatgatcg aagagcgact
     2101 gaagacgtat gcccatctct tcgacgataa agtgatgaaa caactcaaac gccggcggta
     2161 cacgggctgg gggcgcctgt cccgcaagct gatcaacggg atccgggaca aacaaagtgg
     2221 gaaaacgatc ctcgacttcc tcaagtcgga tggcttcgcg aaccggaact tcatgcaact
     2281 catccatgac gacagcctca ccttcaagga agatatccaa aaagcccaag tctccggcca
     2341 gggcgactcc ctgcacgaac atatcgcgaa cctggcgggc tcgcccgcca tcaagaaagg
     2401 catcctccag acggtcaaag tggtcgacga actggtgaaa gtgatggggc ggcataaacc
     2461 cgagaatatc gtcatcgaaa tggcccgcga gaaccaaacc acgcagaaag gccagaaaaa
     2521 tagccgggag cgcatgaagc gcatcgagga aggcatcaag gagctgggca gccagatcct
     2581 caaggagcat ccggtcgaaa acacccaact ccaaaatgag aagctgtatc tctactatct
     2641 gcaaaatggg cgcgatatgt acgtcgatca ggagctcgat atcaaccgcc tctccgacta
     2701 cgatgtggat gcgatcgtcc cccagtcctt cctgaaagat gacagcatcg ataacaaggt
     2761 gctcacccgg tcggacaaaa atcgcgggaa gtccgataat gtcccgtccg aagaagtggt
     2821 gaaaaagatg aagaactact ggcggcaact cctcaacgcg aagctgatca cccagcgcaa
     2881 attcgataac ctcaccaagg ccgagcgggg cgggctctcg gagctcgata aggccggctt
     2941 catcaagcgc cagctggtcg agacgcgcca aatcacgaag cacgtggcgc aaatcctcga
     3001 tagccggatg aataccaagt atgacgagaa tgataaactc atccgggaag tgaaagtgat
     3061 cacgctgaaa agcaaactcg tgtccgactt ccggaaggat ttccaattct ataaagtgcg
     3121 cgaaatcaac aactatcatc acgcgcatga cgcgtacctc aatgccgtcg tgggcacggc
     3181 cctcatcaag aagtacccga agctggaaag cgaattcgtc tacggcgatt acaaagtgta
     3241 cgatgtgcgc aaaatgatcg cgaaatcgga acaagaaatc ggcaaggcga ccgcgaagta
     3301 cttcttctat tcgaatatca tgaatttctt caaaaccgaa atcaccctcg cgaacggcga
     3361 aatccgcaaa cgcccgctca tcgagacgaa cggggagacc ggggaaatcg tgtgggacaa
     3421 ggggcgcgac ttcgccaccg tgcggaaagt cctgtccatg ccgcaagtga atatcgtgaa
     3481 aaaaaccgaa gtgcaaaccg gcggcttctc gaaggaaagc atcctcccca aacgcaacag
     3541 cgataaactg atcgcccgga agaaggactg ggaccccaaa aaatatgggg gcttcgactc
     3601 gccgaccgtg gcgtactcgg tcctcgtggt ggcgaaggtc gaaaaaggga aaagcaaaaa
     3661 actgaaaagc gtcaaggaac tgctcggcat cacgatcatg gagcgctcct ccttcgaaaa
     3721 aaacccgatc gatttcctgg aggcgaaagg ctacaaggag gtcaaaaagg acctgatcat
     3781 caagctcccc aaatatagcc tgttcgaact ggaaaatggc cgcaagcgga tgctggcctc
     3841 cgcgggcgag ctgcaaaagg ggaacgaact cgcgctcccg agcaaatacg tcaacttcct
     3901 ctatctcgcg tcgcattatg aaaagctgaa ggggagcccc gaatataatg aacaaaaaca
     3961 gctgttcgtg gaacaacata aacactatct cgatgagatc atcgagcaaa tctcggagtt
     4021 ctccaaacgg gtcatcctcg cggacgccaa tctcgataag gtgctgtcgg cgtacaataa
     4081 acatcgcgat aagccgatcc gcgagcaagc cgagaacatc atccacctct tcacgctgac
     4141 gaatctgggc gcgccggccg ccttcaagta cttcgacacg acgatcgatc ggaagcggta
     4201 cacctcgacg aaagaagtgc tcgacgccac gctgatccac caatccatca cgggtctgta
     4261 tgaaacccgc atcgacctct cgcaactcgg cggggactaa ctcgagtaag gatctccagg
     4321 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     4381 tcggtgaacg ctctcgggat ccactagttc tagagcggcc gcgtcgtgac tgggaaaacc
     4441 ctggcgacta gtcttggact cctgttgata gatccagtaa tgacctcaga actccatctg
     4501 gatttgttca gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tccagccacg
     4561 cccctcttta atacgacggg caatttgcac ttcagaaaat gaagagtttg ctttagccat
     4621 aacaaaagtc cagtatgctt tttcacagca taactggact gatttcagtt tacaactatt
     4681 ctgtctagtt taagacttta ttgtcatagt ttagatctat tttgttcagt ttaagacttt
     4741 attgtccgcc cacaatttaa atttgtgtct caaaatctct gatgttacat tgcacaagat
     4801 aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     4861 gttatgagcc atattcagcg tgaaacgagc tgtagccgtc cgcgtctgaa cagcaacatg
     4921 gatgcggatc tgtatggcta taaatgggcg cgtgataacg tgggtcagag cggcgcgacc
     4981 atttatcgtc tgtatggcaa accggatgcg ccggaactgt ttctgaaaca tggcaaaggc
     5041 agcgtggcga acgatgtgac cgatgaaatg gtgcgtctga actggctgac cgaatttatg
     5101 ccgctgccga ccattaaaca ttttattcgc accccggatg atgcgtggct gctgaccacc
     5161 gcgattccgg gcaaaaccgc gtttcaggtg ctggaagaat atccggatag cggcgaaaac
     5221 attgtggatg cgctggccgt gtttctgcgt cgtctgcata gcattccggt gtgcaactgc
     5281 ccgtttaaca gcgatcgtgt gtttcgtctg gcccaggcgc agagccgtat gaacaacggc
     5341 ctggtggatg cgagcgattt tgatgatgaa cgtaacggct ggccggtgga acaggtgtgg
     5401 aaagaaatgc ataaactgct gccgtttagc ccggatagcg tggtgaccca cggcgatttt
     5461 agcctggata acctgatttt cgatgaaggc aaactgattg gctgcattga tgtgggccgt
     5521 gtgggcattg cggatcgtta tcaggatctg gccattctgt ggaactgcct gggcgaattt
     5581 agcccgagcc tgcaaaaacg tctgtttcag aaatatggca ttgataatcc ggatatgaac
     5641 aaactgcaat ttcatctgat gctggatgaa tttttctaat aattaattgg accgcggtcc
     5701 gcgcgttgtc cttttccgct gcataaccct gcttcggggt cattatagcg attttttcgg
     5761 tatatccatc ctttttcgca cgatatacag gattttgcca aagggttcgt gtagactttc
     5821 cttggtgtat ccaacggcgt cagccgggca ggataggtga agtaggccca cccgcgagcg
     5881 ggtgttcctt cttcactgtc ccttattcgc acctggcggt gctcaacggg aatcctgctc
     5941 tgcgaggctg gccgtaggcc ggccggcgcg ccccatgtca gccgttaagt gttcctgtgt
     6001 cactcaaaat tgctttgaga ggctctaagg gcttctcagt gcgttacatc cctggcttgt
     6061 tgtccacaac cgttaaacct taaaggcttt aaaagcctta tatattcttt tttttcttat
     6121 aaaacttaaa accttagagg ctatttaagt tgctgattta tattaatttt attgttcaaa
     6181 catgagagct tagtacgtga aacatgagag cttagtacgt tagccatgag agcttagtac
     6241 gttagccatg agggtttagt tcgttaaaca tgagagctta gtacgttaaa catgagagct
     6301 tagtacgtga aacatgagag cttagtacgt actatcaaca ggttgaactg ctgatcttca
     6361 gatcggcgcg cctgtgggcg gacaaaatag ttgggaactg ggaggggtgg aaatggagtt
     6421 tttaaggatt atttagggaa gagtgacaaa atagatggga actgggtgta gcgtcgtaag
     6481 ctaatacgaa aattaaaaat gacaaaatag tttggaacta gatttcactt atctggtttc
     6541 tagggcggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga
     6601 aaggcccagt ctttcgactg agcctttcgt tttatttgat gccgacgtcc ggacgatcga
     6661 attggggatc ttgaagtacc tattccgaag ttcctattct ctagaaagta taggaacttc
     6721 agagcgcttt tgaagctgat gtgcttaaaa acttactcaa tggaattcta gatcgaattg
     6781 gccgcggcgt tgtgacaatt taccgaacaa ctccgcggcc gggaagccga tctcggcttg
     6841 aacgaattgt taggtggcgg tacttgggtc gatatcaaag tgcatcactt cttcccgtat
     6901 gcccaacttt gtatagagag ccactgcggg atcgtcaccg taatctgctt gcacgtagat
     6961 cacataagca ccaagcgcgt tggcctcatg cttgaggaga ttgatgagcg cggtggcaat
     7021 gccctgcctc cggtgctcgc cggagactgc gagatcatag atatagatct cactacgcgg
     7081 ctgctcaaac ttgggcagaa cgtaagccgc gagagcgcca acaaccgctt cttggtcgaa
     7141 ggcagcaagc gcgatgaatg tcttactacg gagcaagttc ccgaggtaat cggagtccgg
     7201 ctgatgttgg gagtaggtgg ctacgtctcc gaactcacga ccgaaaagat caagagcagc
     7261 ccgcatggat ttgacttggt cagggccgag cctacatgtg cgaatgatgc ccatacttga
     7321 gccacctaac tttgttttag ggcgactgcc ctgctgcgta acatcgttgc tgctgcgtaa
     7381 catcgttgct gctccataac atcaaacatc gacccacggc gtaacgcgct tgctgcttgg
     7441 atgcccgagg catagactgt acaaaaaaac agtcataaca agccatgaaa accgccactg
     7501 cgccgttacc accgctgcgt tcggtcaagg ttctggacca gttgcgtgag cgcatacgct
     7561 acttgcatta cagtttacga accgaacagg cttatgtcaa tccgatccat tgctgttgac
     7621 aaagggaatc aggggatctt gaagttccta ttccgaagtt cctattctct agaaagtata
     7681 ggaacttc
//
