LOCUS       sequence-440946-        4228 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini-Tn7 GFP expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4228)
  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 4228)
  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-GFP
FEATURES             Location/Qualifiers
     source          1..4228
                     /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)"
     misc_feature    898..954
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     terminator      997..1091
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1393..1412)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1404..2219
                     /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            2386..2494
                     /note="incP origin of transfer"
     rep_origin      2516..2904
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     mobile_element  2913..3078
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     terminator      complement(3092..3178)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    3213..3260
                     /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(3390..3923)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4112..4117,4118..4134,4135..4140))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    4181..4228
                     /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 gttttctaat gatcatggga
      181 attcataaag gtgaagaact gttcaccggt gttgttccga tcctggttga actggatggt
      241 gatgttaacg gccacaaatt ctctgttcgt ggtgaaggtg aaggtgatgc aaccaacggt
      301 aaactgaccc tgaaattcat ctgcactacc ggtaaactgc cggttccatg gccgactctg
      361 gtgactaccc tgacctatgg tgttcagtgt ttttctcgtt acccggatca catgaagcag
      421 catgatttct tcaaatctgc aatgccggaa ggttatgtac aggagcgcac catttctttc
      481 aaagacgatg gcacctacaa aacccgtgca gaggttaaat ttgaaggtga tactctggtg
      541 aaccgtattg aactgaaagg cattgatttc aaagaggacg gcaacatcct gggccacaaa
      601 ctggaatata acttcaactc ccataacgtt tacatcaccg cagacaaaca gaagaacggt
      661 atcaaagcta acttcaaaat tcgccataac gttgaagacg gtagcgtaca gctggcggac
      721 cactaccagc agaacactcc gatcggtgat ggtccggttc tgctgccgga taaccactac
      781 ctgtccaccc agtctaaact gtccaaagac ccgaacgaaa agcgcgacca catggtgctg
      841 ctggagttcg ttactgcagc aggtatcacg cacggcatgg atgaactcta caaataagaa
      901 ttcgagctcg gtacccgggg atcctctaga gtcgacctgc aggcatgcaa gcttgcggcc
      961 gcgtcgtgac tgggaaaacc ctggcgacta gtcttggact cctgttgata gatccagtaa
     1021 tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg gcgtttttta
     1081 ttggtgagaa tccagccacg cccctcttta atacgacggg caatttgcac ttcagaaaat
     1141 gaagagtttg ctttagccat aacaaaagtc cagtatgctt tttcacagca taactggact
     1201 gatttcagtt tacaactatt ctgtctagtt taagacttta ttgtcatagt ttagatctat
     1261 tttgttcagt ttaagacttt attgtccgcc cacaatttaa atttgtgtct caaaatctct
     1321 gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
     1381 taaacagtaa tacaaggggt gttatgagcc atattcagcg tgaaacgagc tgtagccgtc
     1441 cgcgtctgaa cagcaacatg gatgcggatc tgtatggcta taaatgggcg cgtgataacg
     1501 tgggtcagag cggcgcgacc atttatcgtc tgtatggcaa accggatgcg ccggaactgt
     1561 ttctgaaaca tggcaaaggc agcgtggcga acgatgtgac cgatgaaatg gtgcgtctga
     1621 actggctgac cgaatttatg ccgctgccga ccattaaaca ttttattcgc accccggatg
     1681 atgcgtggct gctgaccacc gcgattccgg gcaaaaccgc gtttcaggtg ctggaagaat
     1741 atccggatag cggcgaaaac attgtggatg cgctggccgt gtttctgcgt cgtctgcata
     1801 gcattccggt gtgcaactgc ccgtttaaca gcgatcgtgt gtttcgtctg gcccaggcgc
     1861 agagccgtat gaacaacggc ctggtggatg cgagcgattt tgatgatgaa cgtaacggct
     1921 ggccggtgga acaggtgtgg aaagaaatgc ataaactgct gccgtttagc ccggatagcg
     1981 tggtgaccca cggcgatttt agcctggata acctgatttt cgatgaaggc aaactgattg
     2041 gctgcattga tgtgggccgt gtgggcattg cggatcgtta tcaggatctg gccattctgt
     2101 ggaactgcct gggcgaattt agcccgagcc tgcaaaaacg tctgtttcag aaatatggca
     2161 ttgataatcc ggatatgaac aaactgcaat ttcatctgat gctggatgaa tttttctaat
     2221 aattaattgg accgcggtcc gcgcgttgtc cttttccgct gcataaccct gcttcggggt
     2281 cattatagcg attttttcgg tatatccatc ctttttcgca cgatatacag gattttgcca
     2341 aagggttcgt gtagactttc cttggtgtat ccaacggcgt cagccgggca ggataggtga
     2401 agtaggccca cccgcgagcg ggtgttcctt cttcactgtc ccttattcgc acctggcggt
     2461 gctcaacggg aatcctgctc tgcgaggctg gccgtaggcc ggccggcgcg ccccatgtca
     2521 gccgttaagt gttcctgtgt cactcaaaat tgctttgaga ggctctaagg gcttctcagt
     2581 gcgttacatc cctggcttgt tgtccacaac cgttaaacct taaaggcttt aaaagcctta
     2641 tatattcttt tttttcttat aaaacttaaa accttagagg ctatttaagt tgctgattta
     2701 tattaatttt attgttcaaa catgagagct tagtacgtga aacatgagag cttagtacgt
     2761 tagccatgag agcttagtac gttagccatg agggtttagt tcgttaaaca tgagagctta
     2821 gtacgttaaa catgagagct tagtacgtga aacatgagag cttagtacgt actatcaaca
     2881 ggttgaactg ctgatcttca gatcggcgcg cctgtgggcg gacaaaatag ttgggaactg
     2941 ggaggggtgg aaatggagtt tttaaggatt atttagggaa gagtgacaaa atagatggga
     3001 actgggtgta gcgtcgtaag ctaatacgaa aattaaaaat gacaaaatag tttggaacta
     3061 gatttcactt atctggtttc tagggcggcg gatttgtcct actcaggaga gcgttcaccg
     3121 acaaacaaca gataaaacga aaggcccagt ctttcgactg agcctttcgt tttatttgat
     3181 gccgacgtcc ggacgatcga attggggatc ttgaagtacc tattccgaag ttcctattct
     3241 ctagaaagta taggaacttc agagcgcttt tgaagctgat gtgcttaaaa acttactcaa
     3301 tggaattcta gatcgaattg gccgcggcgt tgtgacaatt taccgaacaa ctccgcggcc
     3361 gggaagccga tctcggcttg aacgaattgt taggtggcgg tacttgggtc gatatcaaag
     3421 tgcatcactt cttcccgtat gcccaacttt gtatagagag ccactgcggg atcgtcaccg
     3481 taatctgctt gcacgtagat cacataagca ccaagcgcgt tggcctcatg cttgaggaga
     3541 ttgatgagcg cggtggcaat gccctgcctc cggtgctcgc cggagactgc gagatcatag
     3601 atatagatct cactacgcgg ctgctcaaac ttgggcagaa cgtaagccgc gagagcgcca
     3661 acaaccgctt cttggtcgaa ggcagcaagc gcgatgaatg tcttactacg gagcaagttc
     3721 ccgaggtaat cggagtccgg ctgatgttgg gagtaggtgg ctacgtctcc gaactcacga
     3781 ccgaaaagat caagagcagc ccgcatggat ttgacttggt cagggccgag cctacatgtg
     3841 cgaatgatgc ccatacttga gccacctaac tttgttttag ggcgactgcc ctgctgcgta
     3901 acatcgttgc tgctgcgtaa catcgttgct gctccataac atcaaacatc gacccacggc
     3961 gtaacgcgct tgctgcttgg atgcccgagg catagactgt acaaaaaaac agtcataaca
     4021 agccatgaaa accgccactg cgccgttacc accgctgcgt tcggtcaagg ttctggacca
     4081 gttgcgtgag cgcatacgct acttgcatta cagtttacga accgaacagg cttatgtcaa
     4141 tccgatccat tgctgttgac aaagggaatc aggggatctt gaagttccta ttccgaagtt
     4201 cctattctct agaaagtata ggaacttc
//
