LOCUS       sequence-438908-        6167 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini-Tn7 GFP expression vector - XylS/pM promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6167)
  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 6167)
  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-XylS-GFP
FEATURES             Location/Qualifiers
     source          1..6167
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(61..1026)
                     /codon_start=1
                     /gene="xylS"
                     /product="XylS regulator encoded by the Pseudomonas putida 
                     TOL plasmid pWWO"
                     /label=XylS
                     /note="stimulates transcription from the Pm promoter in the
                     presence of aromatic compounds such as m-toluate or 
                     benzoate"
                     /translation="MDFCLLNEKSQIFVHAEPYAVSDYVNQYVGTHSIRLPKGGRPAGR
                     LHHRIFGCLDLCRISYGGSVRVISPGLETCYHLQIILKGHCLWRGHGQEHYFAPGELLL
                     LNPDDQADLTYSEDCEKFIVKLPSVVLDRACSDNNWHKPREGIRFAARHNLQQLDGFIN
                     LLGLVCDEAEHTKSMPRVQEHYAGIIASKLLEMLGSNVSREIFSKGNPSFERVVQFIEE
                     NLKRNISLERLAELAMMSPRSLYNLFEKHAGTTPKNYIRNRKLESIRACLNDPSANVRS
                     ITEIALDYGFLHLGRFAENYRSAFGELPSDTLRQCKKEVA"
     promoter        1926..2006
                     /label=Pm promoter
                     /note="The bacterial Pm promoter is activated by XylS in 
                     the presence of benzoate or m-toluate (Marques et al., 
                     1999)."
     RBS             2112..2120
                     /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)"
     misc_feature    2856..2912
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     terminator      2955..3049
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(3351..3370)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3362..4177
                     /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            4344..4452
                     /note="incP origin of transfer"
     rep_origin      4474..4862
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     mobile_element  4871..5036
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     terminator      complement(5050..5136)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    5171..5218
                     /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(5348..5881)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(6051..6056,6057..6073,6074..6079))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    6120..6167
                     /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 acgttcgtaa
       61 tcaagccact tcctttttgc attgacgcag ggtgtcggaa ggcaactcgc cgaacgcgct
      121 cctatagttt tcagcgaagc gtcccaaatg taagaagccg tagtctaggg ctatctcagt
      181 tatactacgc acattggcac tgggatcgtt caagcaggcg cggatgcttt cgagcttgcg
      241 gttgcggatg tagttcttcg gcgtggtgcc ggcgtgcttc tcgaacaaat tgtagagcga
      301 gcgtggactc atcatcgcca gctccgctaa ccgctcaagg ctgatattcc gtttgagatt
      361 ctcctcaatg aattgaacga ctcgctcgaa agacgggtta cctttgctga aaatttcacg
      421 gctgacattg ctgcccagca tttcgagcag cttggaagcg atgatccccg catagtgctc
      481 ttggacccga ggcatcgact ttgtatgttc cgcttcgtca caaactaacc cgagtagatt
      541 gataaagcca tcgagttgct ggagattgtg tcgcgcggcg aaacggatac cctccctcgg
      601 cttgtgccaa ttgttgtcac tgcacgcccg atcaaggacc actgagggca atttaacgat
      661 aaatttctcg caatcttctg aataggtcag gtcggcttgg tcatccggat tgagcagcaa
      721 tagttcgccc ggcgcaaaat agtgctcctg gccatggcca cgccacaggc aatggccttt
      781 gagtattatt tgcagatgat aacaggtctc taatccaggc gagattaccc tcacgctacc
      841 gccgtagctg attcgacaca gatcgaggca tccgaagatt ctgtggtgca gcctgcctgc
      901 cgggcgcccg cccttgggca ggcgaataga gtgcgtaccg acatactggt taacataatc
      961 ggagactgca tagggctcgg cgtggacgaa gatctgactt ttctcgttca ataagcaaaa
     1021 atccatagtt cacggttctc ttattttaat gtgggctgct tggtgtgatg tagaaaggcg
     1081 ccaagtcgat gaaaatgcat ctcgacgtga tgcgtatacg ggttaccccc attgccacgt
     1141 tgcgccatcc tttttgcaat cagtgaccac ttttccaagc aaaaataacg ccaagcagaa
     1201 cgaagacgtt ctttttaaga agcgagaaca ccagaagttc gtgctgtcgg ggcatggggc
     1261 gacgaattgg cggataaagg ggatctgctg gatattacgg cctttttaaa gaccgtaaag
     1321 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct
     1381 catccgtaat tacgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac
     1441 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac
     1501 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa
     1561 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc
     1621 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc
     1681 gttttcacca tgcatgggaa ttagcttgat ctgaccaacg accggtagcg gagctatcca
     1741 acggcggtat accaggaaaa cacacagcag gtacatcaga acagtaccat gactgaagaa
     1801 caaatagttt tttcctgatc cataaagcag aacggcctgc tccatgacaa atctggctcc
     1861 ccaactaatg ccccatgcag ccagcataac cagcataaag tgcagtgtcc ggtttgatag
     1921 ggataagtcc agccttgcaa gaagcggata caggagtgca aaaaatggct atctctagta
     1981 aggcctaccc cttaggcttt atgcaacaga aacaataata atggagtcat gaccatgcct
     2041 agggcccaag ttcacttaaa aaggagatca acaatgaaag caattttcgt actgaaacat
     2101 cttaatcatg ctaaggaggt tttctaatga tcatgggaat tcataaaggt gaagaactgt
     2161 tcaccggtgt tgttccgatc ctggttgaac tggatggtga tgttaacggc cacaaattct
     2221 ctgttcgtgg tgaaggtgaa ggtgatgcaa ccaacggtaa actgaccctg aaattcatct
     2281 gcactaccgg taaactgccg gttccatggc cgactctggt gactaccctg acctatggtg
     2341 ttcagtgttt ttctcgttac ccggatcaca tgaagcagca tgatttcttc aaatctgcaa
     2401 tgccggaagg ttatgtacag gagcgcacca tttctttcaa agacgatggc acctacaaaa
     2461 cccgtgcaga ggttaaattt gaaggtgata ctctggtgaa ccgtattgaa ctgaaaggca
     2521 ttgatttcaa agaggacggc aacatcctgg gccacaaact ggaatataac ttcaactccc
     2581 ataacgttta catcaccgca gacaaacaga agaacggtat caaagctaac ttcaaaattc
     2641 gccataacgt tgaagacggt agcgtacagc tggcggacca ctaccagcag aacactccga
     2701 tcggtgatgg tccggttctg ctgccggata accactacct gtccacccag tctaaactgt
     2761 ccaaagaccc gaacgaaaag cgcgaccaca tggtgctgct ggagttcgtt actgcagcag
     2821 gtatcacgca cggcatggat gaactctaca aataagaatt cgagctcggt acccggggat
     2881 cctctagagt cgacctgcag gcatgcaagc ttgcggccgc gtcgtgactg ggaaaaccct
     2941 ggcgactagt cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga
     3001 tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc cagccacgcc
     3061 cctctttaat acgacgggca atttgcactt cagaaaatga agagtttgct ttagccataa
     3121 caaaagtcca gtatgctttt tcacagcata actggactga tttcagttta caactattct
     3181 gtctagttta agactttatt gtcatagttt agatctattt tgttcagttt aagactttat
     3241 tgtccgccca caatttaaat ttgtgtctca aaatctctga tgttacattg cacaagataa
     3301 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     3361 tatgagccat attcagcgtg aaacgagctg tagccgtccg cgtctgaaca gcaacatgga
     3421 tgcggatctg tatggctata aatgggcgcg tgataacgtg ggtcagagcg gcgcgaccat
     3481 ttatcgtctg tatggcaaac cggatgcgcc ggaactgttt ctgaaacatg gcaaaggcag
     3541 cgtggcgaac gatgtgaccg atgaaatggt gcgtctgaac tggctgaccg aatttatgcc
     3601 gctgccgacc attaaacatt ttattcgcac cccggatgat gcgtggctgc tgaccaccgc
     3661 gattccgggc aaaaccgcgt ttcaggtgct ggaagaatat ccggatagcg gcgaaaacat
     3721 tgtggatgcg ctggccgtgt ttctgcgtcg tctgcatagc attccggtgt gcaactgccc
     3781 gtttaacagc gatcgtgtgt ttcgtctggc ccaggcgcag agccgtatga acaacggcct
     3841 ggtggatgcg agcgattttg atgatgaacg taacggctgg ccggtggaac aggtgtggaa
     3901 agaaatgcat aaactgctgc cgtttagccc ggatagcgtg gtgacccacg gcgattttag
     3961 cctggataac ctgattttcg atgaaggcaa actgattggc tgcattgatg tgggccgtgt
     4021 gggcattgcg gatcgttatc aggatctggc cattctgtgg aactgcctgg gcgaatttag
     4081 cccgagcctg caaaaacgtc tgtttcagaa atatggcatt gataatccgg atatgaacaa
     4141 actgcaattt catctgatgc tggatgaatt tttctaataa ttaattggac cgcggtccgc
     4201 gcgttgtcct tttccgctgc ataaccctgc ttcggggtca ttatagcgat tttttcggta
     4261 tatccatcct ttttcgcacg atatacagga ttttgccaaa gggttcgtgt agactttcct
     4321 tggtgtatcc aacggcgtca gccgggcagg ataggtgaag taggcccacc cgcgagcggg
     4381 tgttccttct tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa tcctgctctg
     4441 cgaggctggc cgtaggccgg ccggcgcgcc ccatgtcagc cgttaagtgt tcctgtgtca
     4501 ctcaaaattg ctttgagagg ctctaagggc ttctcagtgc gttacatccc tggcttgttg
     4561 tccacaaccg ttaaacctta aaggctttaa aagccttata tattcttttt tttcttataa
     4621 aacttaaaac cttagaggct atttaagttg ctgatttata ttaattttat tgttcaaaca
     4681 tgagagctta gtacgtgaaa catgagagct tagtacgtta gccatgagag cttagtacgt
     4741 tagccatgag ggtttagttc gttaaacatg agagcttagt acgttaaaca tgagagctta
     4801 gtacgtgaaa catgagagct tagtacgtac tatcaacagg ttgaactgct gatcttcaga
     4861 tcggcgcgcc tgtgggcgga caaaatagtt gggaactggg aggggtggaa atggagtttt
     4921 taaggattat ttagggaaga gtgacaaaat agatgggaac tgggtgtagc gtcgtaagct
     4981 aatacgaaaa ttaaaaatga caaaatagtt tggaactaga tttcacttat ctggtttcta
     5041 gggcggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa
     5101 ggcccagtct ttcgactgag cctttcgttt tatttgatgc cgacgtccgg acgatcgaat
     5161 tggggatctt gaagtaccta ttccgaagtt cctattctct agaaagtata ggaacttcag
     5221 agcgcttttg aagctgatgt gcttaaaaac ttactcaatg gaattctaga tcgaattggc
     5281 cgcggcgttg tgacaattta ccgaacaact ccgcggccgg gaagccgatc tcggcttgaa
     5341 cgaattgtta ggtggcggta cttgggtcga tatcaaagtg catcacttct tcccgtatgc
     5401 ccaactttgt atagagagcc actgcgggat cgtcaccgta atctgcttgc acgtagatca
     5461 cataagcacc aagcgcgttg gcctcatgct tgaggagatt gatgagcgcg gtggcaatgc
     5521 cctgcctccg gtgctcgccg gagactgcga gatcatagat atagatctca ctacgcggct
     5581 gctcaaactt gggcagaacg taagccgcga gagcgccaac aaccgcttct tggtcgaagg
     5641 cagcaagcgc gatgaatgtc ttactacgga gcaagttccc gaggtaatcg gagtccggct
     5701 gatgttggga gtaggtggct acgtctccga actcacgacc gaaaagatca agagcagccc
     5761 gcatggattt gacttggtca gggccgagcc tacatgtgcg aatgatgccc atacttgagc
     5821 cacctaactt tgttttaggg cgactgccct gctgcgtaac atcgttgctg ctccataaca
     5881 tcaaacatcg acccacggcg taacgcgctt gctgcttgga tgcccgaggc atagactgta
     5941 caaaaaaaca gtcataacaa gccatgaaaa ccgccactgc gccgttacca ccgctgcgtt
     6001 cggtcaaggt tctggaccag ttgcgtgagc gcatacgcta cttgcattac agtttacgaa
     6061 ccgaacaggc ttatgtcaat tcgatccatt gctgttgaca aagggaatca ggggatcttg
     6121 aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttc
//
