LOCUS       sequence-438907-        5815 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini-Tn7 GFP expression vector - LacI/pLac promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5815)
  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 5815)
  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-LacI-GFP
FEATURES             Location/Qualifiers
     source          1..5815
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(82..1164)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     1126..1145
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1165..1242)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     protein_bind    1615..1631
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             1741..1749
                     /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    2485..2541
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     terminator      2584..2678
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2980..2999)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2991..3806
                     /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            3973..4081
                     /note="incP origin of transfer"
     rep_origin      4103..4491
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     mobile_element  4500..4665
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     terminator      complement(4679..4765)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4800..4847
                     /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(4977..5510)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(5699..5704,5705..5721,5722..5727))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    5768..5815
                     /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 agtgagctaa
       61 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
      121 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
      181 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
      241 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
      301 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
      361 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
      421 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
      481 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
      541 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
      601 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
      661 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
      721 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
      781 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
      841 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
      901 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
      961 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     1021 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     1081 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     1141 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     1201 tcatgccata ccgcgaaagg ttttgcacca ttcgatggtg tcaacgtaaa tgcatgccgc
     1261 ttgggaggca gataggtagg catggccccc attttcaata caagcaacgc atgagaaagc
     1321 ccccggaaga tcaccttccg ggggcttttt tattgcgctc cggcaattaa aaaagcggct
     1381 aaccacgccg ctttttttac gtctgcaggc gaattgatct ggtttgacag cttatcacca
     1441 agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc
     1501 aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa
     1561 gatcatctta ttaatcagat aaaatattat aaatgtgagc ggataacatt gacattgtga
     1621 gcggataaca agatactgag cacatcagca ggtttcacac aggaaaccta gggcccaagt
     1681 tcacttaaaa aggagatcaa caatgaaagc aattttcgta ctgaaacatc ttaatcatgc
     1741 taaggaggtt ttctaatgat catgggaatt cataaaggtg aagaactgtt caccggtgtt
     1801 gttccgatcc tggttgaact ggatggtgat gttaacggcc acaaattctc tgttcgtggt
     1861 gaaggtgaag gtgatgcaac caacggtaaa ctgaccctga aattcatctg cactaccggt
     1921 aaactgccgg ttccatggcc gactctggtg actaccctga cctatggtgt tcagtgtttt
     1981 tctcgttacc cggatcacat gaagcagcat gatttcttca aatctgcaat gccggaaggt
     2041 tatgtacagg agcgcaccat ttctttcaaa gacgatggca cctacaaaac ccgtgcagag
     2101 gttaaatttg aaggtgatac tctggtgaac cgtattgaac tgaaaggcat tgatttcaaa
     2161 gaggacggca acatcctggg ccacaaactg gaatataact tcaactccca taacgtttac
     2221 atcaccgcag acaaacagaa gaacggtatc aaagctaact tcaaaattcg ccataacgtt
     2281 gaagacggta gcgtacagct ggcggaccac taccagcaga acactccgat cggtgatggt
     2341 ccggttctgc tgccggataa ccactacctg tccacccagt ctaaactgtc caaagacccg
     2401 aacgaaaagc gcgaccacat ggtgctgctg gagttcgtta ctgcagcagg tatcacgcac
     2461 ggcatggatg aactctacaa ataagaattc gagctcggta cccggggatc ctctagagtc
     2521 gacctgcagg catgcaagct tgcggccgcg tcgtgactgg gaaaaccctg gcgactagtc
     2581 ttggactcct gttgatagat ccagtaatga cctcagaact ccatctggat ttgttcagaa
     2641 cgctcggttg ccgccgggcg ttttttattg gtgagaatcc agccacgccc ctctttaata
     2701 cgacgggcaa tttgcacttc agaaaatgaa gagtttgctt tagccataac aaaagtccag
     2761 tatgcttttt cacagcataa ctggactgat ttcagtttac aactattctg tctagtttaa
     2821 gactttattg tcatagttta gatctatttt gttcagttta agactttatt gtccgcccac
     2881 aatttaaatt tgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat
     2941 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
     3001 ttcagcgtga aacgagctgt agccgtccgc gtctgaacag caacatggat gcggatctgt
     3061 atggctataa atgggcgcgt gataacgtgg gtcagagcgg cgcgaccatt tatcgtctgt
     3121 atggcaaacc ggatgcgccg gaactgtttc tgaaacatgg caaaggcagc gtggcgaacg
     3181 atgtgaccga tgaaatggtg cgtctgaact ggctgaccga atttatgccg ctgccgacca
     3241 ttaaacattt tattcgcacc ccggatgatg cgtggctgct gaccaccgcg attccgggca
     3301 aaaccgcgtt tcaggtgctg gaagaatatc cggatagcgg cgaaaacatt gtggatgcgc
     3361 tggccgtgtt tctgcgtcgt ctgcatagca ttccggtgtg caactgcccg tttaacagcg
     3421 atcgtgtgtt tcgtctggcc caggcgcaga gccgtatgaa caacggcctg gtggatgcga
     3481 gcgattttga tgatgaacgt aacggctggc cggtggaaca ggtgtggaaa gaaatgcata
     3541 aactgctgcc gtttagcccg gatagcgtgg tgacccacgg cgattttagc ctggataacc
     3601 tgattttcga tgaaggcaaa ctgattggct gcattgatgt gggccgtgtg ggcattgcgg
     3661 atcgttatca ggatctggcc attctgtgga actgcctggg cgaatttagc ccgagcctgc
     3721 aaaaacgtct gtttcagaaa tatggcattg ataatccgga tatgaacaaa ctgcaatttc
     3781 atctgatgct ggatgaattt ttctaataat taattggacc gcggtccgcg cgttgtcctt
     3841 ttccgctgca taaccctgct tcggggtcat tatagcgatt ttttcggtat atccatcctt
     3901 tttcgcacga tatacaggat tttgccaaag ggttcgtgta gactttcctt ggtgtatcca
     3961 acggcgtcag ccgggcagga taggtgaagt aggcccaccc gcgagcgggt gttccttctt
     4021 cactgtccct tattcgcacc tggcggtgct caacgggaat cctgctctgc gaggctggcc
     4081 gtaggccggc cggcgcgccc catgtcagcc gttaagtgtt cctgtgtcac tcaaaattgc
     4141 tttgagaggc tctaagggct tctcagtgcg ttacatccct ggcttgttgt ccacaaccgt
     4201 taaaccttaa aggctttaaa agccttatat attctttttt ttcttataaa acttaaaacc
     4261 ttagaggcta tttaagttgc tgatttatat taattttatt gttcaaacat gagagcttag
     4321 tacgtgaaac atgagagctt agtacgttag ccatgagagc ttagtacgtt agccatgagg
     4381 gtttagttcg ttaaacatga gagcttagta cgttaaacat gagagcttag tacgtgaaac
     4441 atgagagctt agtacgtact atcaacaggt tgaactgctg atcttcagat cggcgcgcct
     4501 gtgggcggac aaaatagttg ggaactggga ggggtggaaa tggagttttt aaggattatt
     4561 tagggaagag tgacaaaata gatgggaact gggtgtagcg tcgtaagcta atacgaaaat
     4621 taaaaatgac aaaatagttt ggaactagat ttcacttatc tggtttctag ggcggcggat
     4681 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     4741 tcgactgagc ctttcgtttt atttgatgcc gacgtccgga cgatcgaatt ggggatcttg
     4801 aagtacctat tccgaagttc ctattctcta gaaagtatag gaacttcaga gcgcttttga
     4861 agctgatgtg cttaaaaact tactcaatgg aattctagat cgaattggcc gcggcgttgt
     4921 gacaatttac cgaacaactc cgcggccggg aagccgatct cggcttgaac gaattgttag
     4981 gtggcggtac ttgggtcgat atcaaagtgc atcacttctt cccgtatgcc caactttgta
     5041 tagagagcca ctgcgggatc gtcaccgtaa tctgcttgca cgtagatcac ataagcacca
     5101 agcgcgttgg cctcatgctt gaggagattg atgagcgcgg tggcaatgcc ctgcctccgg
     5161 tgctcgccgg agactgcgag atcatagata tagatctcac tacgcggctg ctcaaacttg
     5221 ggcagaacgt aagccgcgag agcgccaaca accgcttctt ggtcgaaggc agcaagcgcg
     5281 atgaatgtct tactacggag caagttcccg aggtaatcgg agtccggctg atgttgggag
     5341 taggtggcta cgtctccgaa ctcacgaccg aaaagatcaa gagcagcccg catggatttg
     5401 acttggtcag ggccgagcct acatgtgcga atgatgccca tacttgagcc acctaacttt
     5461 gttttagggc gactgccctg ctgcgtaaca tcgttgctgc tgcgtaacat cgttgctgct
     5521 ccataacatc aaacatcgac ccacggcgta acgcgcttgc tgcttggatg cccgaggcat
     5581 agactgtaca aaaaaacagt cataacaagc catgaaaacc gccactgcgc cgttaccacc
     5641 gctgcgttcg gtcaaggttc tggaccagtt gcgtgagcgc atacgctact tgcattacag
     5701 tttacgaacc gaacaggctt atgtcaatcc gatccattgc tgttgacaaa gggaatcagg
     5761 ggatcttgaa gttcctattc cgaagttcct attctctaga aagtatagga acttc
//
