LOCUS       sequence-440947-        5393 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini-Tn7 GFP expression vector - AraC/pBAD promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5393)
  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 5393)
  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-GFP
FEATURES             Location/Qualifiers
     source          1..5393
                     /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)"
     misc_feature    2063..2119
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     terminator      2162..2256
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2558..2577)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2569..3384
                     /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            3551..3659
                     /note="incP origin of transfer"
     rep_origin      3681..4069
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     mobile_element  4078..4243
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     terminator      complement(4257..4343)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4378..4425
                     /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(4555..5088)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(5277..5282,5283..5299,5300..5305))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    5346..5393
                     /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 aggaggtttt ctaatgatca tgggaattca taaaggtgaa gaactgttca ccggtgttgt
     1381 tccgatcctg gttgaactgg atggtgatgt taacggccac aaattctctg ttcgtggtga
     1441 aggtgaaggt gatgcaacca acggtaaact gaccctgaaa ttcatctgca ctaccggtaa
     1501 actgccggtt ccatggccga ctctggtgac taccctgacc tatggtgttc agtgtttttc
     1561 tcgttacccg gatcacatga agcagcatga tttcttcaaa tctgcaatgc cggaaggtta
     1621 tgtacaggag cgcaccattt ctttcaaaga cgatggcacc tacaaaaccc gtgcagaggt
     1681 taaatttgaa ggtgatactc tggtgaaccg tattgaactg aaaggcattg atttcaaaga
     1741 ggacggcaac atcctgggcc acaaactgga atataacttc aactcccata acgtttacat
     1801 caccgcagac aaacagaaga acggtatcaa agctaacttc aaaattcgcc ataacgttga
     1861 agacggtagc gtacagctgg cggaccacta ccagcagaac actccgatcg gtgatggtcc
     1921 ggttctgctg ccggataacc actacctgtc cacccagtct aaactgtcca aagacccgaa
     1981 cgaaaagcgc gaccacatgg tgctgctgga gttcgttact gcagcaggta tcacgcacgg
     2041 catggatgaa ctctacaaat aagaattcga gctcggtacc cggggatcct ctagagtcga
     2101 cctgcaggca tgcaagcttg cggccgcgtc gtgactggga aaaccctggc gactagtctt
     2161 ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg
     2221 ctcggttgcc gccgggcgtt ttttattggt gagaatccag ccacgcccct ctttaatacg
     2281 acgggcaatt tgcacttcag aaaatgaaga gtttgcttta gccataacaa aagtccagta
     2341 tgctttttca cagcataact ggactgattt cagtttacaa ctattctgtc tagtttaaga
     2401 ctttattgtc atagtttaga tctattttgt tcagtttaag actttattgt ccgcccacaa
     2461 tttaaatttg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     2521 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     2581 cagcgtgaaa cgagctgtag ccgtccgcgt ctgaacagca acatggatgc ggatctgtat
     2641 ggctataaat gggcgcgtga taacgtgggt cagagcggcg cgaccattta tcgtctgtat
     2701 ggcaaaccgg atgcgccgga actgtttctg aaacatggca aaggcagcgt ggcgaacgat
     2761 gtgaccgatg aaatggtgcg tctgaactgg ctgaccgaat ttatgccgct gccgaccatt
     2821 aaacatttta ttcgcacccc ggatgatgcg tggctgctga ccaccgcgat tccgggcaaa
     2881 accgcgtttc aggtgctgga agaatatccg gatagcggcg aaaacattgt ggatgcgctg
     2941 gccgtgtttc tgcgtcgtct gcatagcatt ccggtgtgca actgcccgtt taacagcgat
     3001 cgtgtgtttc gtctggccca ggcgcagagc cgtatgaaca acggcctggt ggatgcgagc
     3061 gattttgatg atgaacgtaa cggctggccg gtggaacagg tgtggaaaga aatgcataaa
     3121 ctgctgccgt ttagcccgga tagcgtggtg acccacggcg attttagcct ggataacctg
     3181 attttcgatg aaggcaaact gattggctgc attgatgtgg gccgtgtggg cattgcggat
     3241 cgttatcagg atctggccat tctgtggaac tgcctgggcg aatttagccc gagcctgcaa
     3301 aaacgtctgt ttcagaaata tggcattgat aatccggata tgaacaaact gcaatttcat
     3361 ctgatgctgg atgaattttt ctaataatta attggaccgc ggtccgcgcg ttgtcctttt
     3421 ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt
     3481 tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac
     3541 ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca
     3601 ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgt
     3661 aggccggccg gcgcgcccca tgtcagccgt taagtgttcc tgtgtcactc aaaattgctt
     3721 tgagaggctc taagggcttc tcagtgcgtt acatccctgg cttgttgtcc acaaccgtta
     3781 aaccttaaag gctttaaaag ccttatatat tctttttttt cttataaaac ttaaaacctt
     3841 agaggctatt taagttgctg atttatatta attttattgt tcaaacatga gagcttagta
     3901 cgtgaaacat gagagcttag tacgttagcc atgagagctt agtacgttag ccatgagggt
     3961 ttagttcgtt aaacatgaga gcttagtacg ttaaacatga gagcttagta cgtgaaacat
     4021 gagagcttag tacgtactat caacaggttg aactgctgat cttcagatcg gcgcgcctgt
     4081 gggcggacaa aatagttggg aactgggagg ggtggaaatg gagtttttaa ggattattta
     4141 gggaagagtg acaaaataga tgggaactgg gtgtagcgtc gtaagctaat acgaaaatta
     4201 aaaatgacaa aatagtttgg aactagattt cacttatctg gtttctaggg cggcggattt
     4261 gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc
     4321 gactgagcct ttcgttttat ttgatgccga cgtccggacg atcgaattgg ggatcttgaa
     4381 gtacctattc cgaagttcct attctctaga aagtatagga acttcagagc gcttttgaag
     4441 ctgatgtgct taaaaactta ctcaatggaa ttctagatcg aattggccgc ggcgttgtga
     4501 caatttaccg aacaactccg cggccgggaa gccgatctcg gcttgaacga attgttaggt
     4561 ggcggtactt gggtcgatat caaagtgcat cacttcttcc cgtatgccca actttgtata
     4621 gagagccact gcgggatcgt caccgtaatc tgcttgcacg tagatcacat aagcaccaag
     4681 cgcgttggcc tcatgcttga ggagattgat gagcgcggtg gcaatgccct gcctccggtg
     4741 ctcgccggag actgcgagat catagatata gatctcacta cgcggctgct caaacttggg
     4801 cagaacgtaa gccgcgagag cgccaacaac cgcttcttgg tcgaaggcag caagcgcgat
     4861 gaatgtctta ctacggagca agttcccgag gtaatcggag tccggctgat gttgggagta
     4921 ggtggctacg tctccgaact cacgaccgaa aagatcaaga gcagcccgca tggatttgac
     4981 ttggtcaggg ccgagcctac atgtgcgaat gatgcccata cttgagccac ctaactttgt
     5041 tttagggcga ctgccctgct gcgtaacatc gttgctgctg cgtaacatcg ttgctgctcc
     5101 ataacatcaa acatcgaccc acggcgtaac gcgcttgctg cttggatgcc cgaggcatag
     5161 actgtacaaa aaaacagtca taacaagcca tgaaaaccgc cactgcgccg ttaccaccgc
     5221 tgcgttcggt caaggttctg gaccagttgc gtgagcgcat acgctacttg cattacagtt
     5281 tacgaaccga acaggcttat gtcaatccga tccattgctg ttgacaaagg gaatcagggg
     5341 atcttgaagt tcctattccg aagttcctat tctctagaaa gtataggaac ttc
//
