LOCUS       sequence-438906-        9275 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini-Tn7 dCas9 expression vector - LacI/pLac promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9275)
  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 9275)
  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-dCas9
FEATURES             Location/Qualifiers
     source          1..9275
                     /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)"
     terminator      5911..5982
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(5985..6001)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5985..6001)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      6044..6138
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(6440..6459)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6451..7266
                     /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            7433..7541
                     /note="incP origin of transfer"
     rep_origin      7563..7951
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     mobile_element  7960..8125
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     terminator      complement(8139..8225)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    8260..8307
                     /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(8437..8970)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(9159..9164,9165..9181,9182..9187))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    9228..9275
                     /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 taaggaggta ccgctagcgg ttatagaagg agatatacat atggataaaa agtacagcat
     1801 cgggctcgcg atcgggacca actccgtggg gtgggccgtc atcacggacg agtataaggt
     1861 cccgtcgaag aaattcaagg tcctcgggaa cacggatcgg catagcatca aaaaagacct
     1921 gatcggggcc ctcctgttcg actccggcga gaccgcggag gcgacgcgcc tcaagcgcac
     1981 ggcccggcgc cgctatacgc ggcgcaaaaa tcggatctgc tatctccaag agatcttcag
     2041 caatgagatg gcgaaagtgg atgactcctt cttccaccgg ctcgaagagt cgttcctggt
     2101 cgaggaagat aaaaagcatg agagccatcc gatcttcggg aatatcgtgg acgaggtggc
     2161 ctaccatgag aaatacccga ccatctatca tctgcggaag aaactggtgg actccaccga
     2221 caaggcggac ctccgcctga tctatctcgc gctggcccat atgatcaagt tccgcgggca
     2281 cttcctgatc gagggggatc tcaaccccga taactcggat gtggacaaac tgttcatcca
     2341 actcgtccag acctataatc aactgttcga agagaatccg atcaatgcca gcggcgtcga
     2401 tgccaaggcg atcctctcgg cccggctctc caagtcgcgg cggctcgaaa atctgatcgc
     2461 ccaactcccc ggcgagaaga agaatggcct gttcgggaac ctcatcgccc tctccctggg
     2521 gctgaccccc aatttcaaaa gcaacttcga cctggccgaa gatgcgaaac tccagctgtc
     2581 caaggatacc tacgacgacg atctggacaa cctcctggcg caaatcgggg atcagtatgc
     2641 cgatctcttc ctggcggcca aaaatctctc cgatgcgatc ctcctgtcgg atatcctgcg
     2701 ggtgaatacc gagatcacca aagcccccct gagcgcctcg atgatcaaac gctacgacga
     2761 gcatcatcaa gacctgaccc tcctgaaggc cctcgtgcgc cagcagctgc cggagaaata
     2821 taaagaaatc ttcttcgatc agtccaaaaa tgggtatgcg gggtacatcg acggcggggc
     2881 ctcgcaggaa gagttctata agttcatcaa accgatcctg gaaaaaatgg atgggaccga
     2941 agagctcctg gtgaaactca accgcgaaga cctcctgcgg aaacagcgca cgttcgacaa
     3001 cggctccatc ccccatcaaa tccacctcgg ggagctccat gcgatcctcc gccggcagga
     3061 agatttctac cccttcctca aagataaccg ggaaaaaatc gagaaaatcc tcaccttccg
     3121 gatcccctac tacgtcggcc cgctggcgcg ggggaactcc cggttcgcgt ggatgacgcg
     3181 caagtcggag gagacgatca ccccctggaa tttcgaagag gtggtggata agggcgcctc
     3241 ggcgcaaagc ttcatcgaac ggatgacgaa tttcgacaaa aacctcccga atgagaaagt
     3301 cctgcccaag cattcgctgc tctacgaata tttcaccgtg tacaatgaac tgacgaaagt
     3361 caagtatgtc accgagggca tgcgcaaacc cgccttcctg tcgggggaac agaaaaaggc
     3421 gatcgtggat ctgctgttca aaaccaatcg caaggtcacg gtgaagcagc tcaaagaaga
     3481 ctatttcaaa aaaatcgagt gcttcgattc cgtggagatc tccggcgtgg aagatcggtt
     3541 caacgcgtcg ctggggacct atcatgatct gctgaaaatc atcaaagata aggacttcct
     3601 ggacaacgaa gaaaatgaag acatcctgga ggacatcgtc ctcacgctga cgctgttcga
     3661 ggatcgcgag atgatcgaag agcgactgaa gacgtatgcc catctcttcg acgataaagt
     3721 gatgaaacaa ctcaaacgcc ggcggtacac gggctggggg cgcctgtccc gcaagctgat
     3781 caacgggatc cgggacaaac aaagtgggaa aacgatcctc gacttcctca agtcggatgg
     3841 cttcgcgaac cggaacttca tgcaactcat ccatgacgac agcctcacct tcaaggaaga
     3901 tatccaaaaa gcccaagtct ccggccaggg cgactccctg cacgaacata tcgcgaacct
     3961 ggcgggctcg cccgccatca agaaaggcat cctccagacg gtcaaagtgg tcgacgaact
     4021 ggtgaaagtg atggggcggc ataaacccga gaatatcgtc atcgaaatgg cccgcgagaa
     4081 ccaaaccacg cagaaaggcc agaaaaatag ccgggagcgc atgaagcgca tcgaggaagg
     4141 catcaaggag ctgggcagcc agatcctcaa ggagcatccg gtcgaaaaca cccaactcca
     4201 aaatgagaag ctgtatctct actatctgca aaatgggcgc gatatgtacg tcgatcagga
     4261 gctcgatatc aaccgcctct ccgactacga tgtggatgcg atcgtccccc agtccttcct
     4321 gaaagatgac agcatcgata acaaggtgct cacccggtcg gacaaaaatc gcgggaagtc
     4381 cgataatgtc ccgtccgaag aagtggtgaa aaagatgaag aactactggc ggcaactcct
     4441 caacgcgaag ctgatcaccc agcgcaaatt cgataacctc accaaggccg agcggggcgg
     4501 gctctcggag ctcgataagg ccggcttcat caagcgccag ctggtcgaga cgcgccaaat
     4561 cacgaagcac gtggcgcaaa tcctcgatag ccggatgaat accaagtatg acgagaatga
     4621 taaactcatc cgggaagtga aagtgatcac gctgaaaagc aaactcgtgt ccgacttccg
     4681 gaaggatttc caattctata aagtgcgcga aatcaacaac tatcatcacg cgcatgacgc
     4741 gtacctcaat gccgtcgtgg gcacggccct catcaagaag tacccgaagc tggaaagcga
     4801 attcgtctac ggcgattaca aagtgtacga tgtgcgcaaa atgatcgcga aatcggaaca
     4861 agaaatcggc aaggcgaccg cgaagtactt cttctattcg aatatcatga atttcttcaa
     4921 aaccgaaatc accctcgcga acggcgaaat ccgcaaacgc ccgctcatcg agacgaacgg
     4981 ggagaccggg gaaatcgtgt gggacaaggg gcgcgacttc gccaccgtgc ggaaagtcct
     5041 gtccatgccg caagtgaata tcgtgaaaaa aaccgaagtg caaaccggcg gcttctcgaa
     5101 ggaaagcatc ctccccaaac gcaacagcga taaactgatc gcccggaaga aggactggga
     5161 ccccaaaaaa tatgggggct tcgactcgcc gaccgtggcg tactcggtcc tcgtggtggc
     5221 gaaggtcgaa aaagggaaaa gcaaaaaact gaaaagcgtc aaggaactgc tcggcatcac
     5281 gatcatggag cgctcctcct tcgaaaaaaa cccgatcgat ttcctggagg cgaaaggcta
     5341 caaggaggtc aaaaaggacc tgatcatcaa gctccccaaa tatagcctgt tcgaactgga
     5401 aaatggccgc aagcggatgc tggcctccgc gggcgagctg caaaagggga acgaactcgc
     5461 gctcccgagc aaatacgtca acttcctcta tctcgcgtcg cattatgaaa agctgaaggg
     5521 gagccccgaa tataatgaac aaaaacagct gttcgtggaa caacataaac actatctcga
     5581 tgagatcatc gagcaaatct cggagttctc caaacgggtc atcctcgcgg acgccaatct
     5641 cgataaggtg ctgtcggcgt acaataaaca tcgcgataag ccgatccgcg agcaagccga
     5701 gaacatcatc cacctcttca cgctgacgaa tctgggcgcg ccggccgcct tcaagtactt
     5761 cgacacgacg atcgatcgga agcggtacac ctcgacgaaa gaagtgctcg acgccacgct
     5821 gatccaccaa tccatcacgg gtctgtatga aacccgcatc gacctctcgc aactcggcgg
     5881 ggactaactc gagtaaggat ctccaggcat caaataaaac gaaaggctca gtcgaaagac
     5941 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcgggatcca ctagttctag
     6001 agcggccgcg tcgtgactgg gaaaaccctg gcgactagtc ttggactcct gttgatagat
     6061 ccagtaatga cctcagaact ccatctggat ttgttcagaa cgctcggttg ccgccgggcg
     6121 ttttttattg gtgagaatcc agccacgccc ctctttaata cgacgggcaa tttgcacttc
     6181 agaaaatgaa gagtttgctt tagccataac aaaagtccag tatgcttttt cacagcataa
     6241 ctggactgat ttcagtttac aactattctg tctagtttaa gactttattg tcatagttta
     6301 gatctatttt gttcagttta agactttatt gtccgcccac aatttaaatt tgtgtctcaa
     6361 aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct
     6421 gcttacataa acagtaatac aaggggtgtt atgagccata ttcagcgtga aacgagctgt
     6481 agccgtccgc gtctgaacag caacatggat gcggatctgt atggctataa atgggcgcgt
     6541 gataacgtgg gtcagagcgg cgcgaccatt tatcgtctgt atggcaaacc ggatgcgccg
     6601 gaactgtttc tgaaacatgg caaaggcagc gtggcgaacg atgtgaccga tgaaatggtg
     6661 cgtctgaact ggctgaccga atttatgccg ctgccgacca ttaaacattt tattcgcacc
     6721 ccggatgatg cgtggctgct gaccaccgcg attccgggca aaaccgcgtt tcaggtgctg
     6781 gaagaatatc cggatagcgg cgaaaacatt gtggatgcgc tggccgtgtt tctgcgtcgt
     6841 ctgcatagca ttccggtgtg caactgcccg tttaacagcg atcgtgtgtt tcgtctggcc
     6901 caggcgcaga gccgtatgaa caacggcctg gtggatgcga gcgattttga tgatgaacgt
     6961 aacggctggc cggtggaaca ggtgtggaaa gaaatgcata aactgctgcc gtttagcccg
     7021 gatagcgtgg tgacccacgg cgattttagc ctggataacc tgattttcga tgaaggcaaa
     7081 ctgattggct gcattgatgt gggccgtgtg ggcattgcgg atcgttatca ggatctggcc
     7141 attctgtgga actgcctggg cgaatttagc ccgagcctgc aaaaacgtct gtttcagaaa
     7201 tatggcattg ataatccgga tatgaacaaa ctgcaatttc atctgatgct ggatgaattt
     7261 ttctaataat taattggacc gcggtccgcg cgttgtcctt ttccgctgca taaccctgct
     7321 tcggggtcat tatagcgatt ttttcggtat atccatcctt tttcgcacga tatacaggat
     7381 tttgccaaag ggttcgtgta gactttcctt ggtgtatcca acggcgtcag ccgggcagga
     7441 taggtgaagt aggcccaccc gcgagcgggt gttccttctt cactgtccct tattcgcacc
     7501 tggcggtgct caacgggaat cctgctctgc gaggctggcc gtaggccggc cggcgcgccc
     7561 catgtcagcc gttaagtgtt cctgtgtcac tcaaaattgc tttgagaggc tctaagggct
     7621 tctcagtgcg ttacatccct ggcttgttgt ccacaaccgt taaaccttaa aggctttaaa
     7681 agccttatat attctttttt ttcttataaa acttaaaacc ttagaggcta tttaagttgc
     7741 tgatttatat taattttatt gttcaaacat gagagcttag tacgtgaaac atgagagctt
     7801 agtacgttag ccatgagagc ttagtacgtt agccatgagg gtttagttcg ttaaacatga
     7861 gagcttagta cgttaaacat gagagcttag tacgtgaaac atgagagctt agtacgtact
     7921 atcaacaggt tgaactgctg atcttcagat cggcgcgcct gtgggcggac aaaatagttg
     7981 ggaactggga ggggtggaaa tggagttttt aaggattatt tagggaagag tgacaaaata
     8041 gatgggaact gggtgtagcg tcgtaagcta atacgaaaat taaaaatgac aaaatagttt
     8101 ggaactagat ttcacttatc tggtttctag ggcggcggat ttgtcctact caggagagcg
     8161 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt
     8221 atttgatgcc gacgtccgga cgatcgaatt ggggatcttg aagtacctat tccgaagttc
     8281 ctattctcta gaaagtatag gaacttcaga gcgcttttga agctgatgtg cttaaaaact
     8341 tactcaatgg aattctagat cgaattggcc gcggcgttgt gacaatttac cgaacaactc
     8401 cgcggccggg aagccgatct cggcttgaac gaattgttag gtggcggtac ttgggtcgat
     8461 atcaaagtgc atcacttctt cccgtatgcc caactttgta tagagagcca ctgcgggatc
     8521 gtcaccgtaa tctgcttgca cgtagatcac ataagcacca agcgcgttgg cctcatgctt
     8581 gaggagattg atgagcgcgg tggcaatgcc ctgcctccgg tgctcgccgg agactgcgag
     8641 atcatagata tagatctcac tacgcggctg ctcaaacttg ggcagaacgt aagccgcgag
     8701 agcgccaaca accgcttctt ggtcgaaggc agcaagcgcg atgaatgtct tactacggag
     8761 caagttcccg aggtaatcgg agtccggctg atgttgggag taggtggcta cgtctccgaa
     8821 ctcacgaccg aaaagatcaa gagcagcccg catggatttg acttggtcag ggccgagcct
     8881 acatgtgcga atgatgccca tacttgagcc acctaacttt gttttagggc gactgccctg
     8941 ctgcgtaaca tcgttgctgc tgcgtaacat cgttgctgct ccataacatc aaacatcgac
     9001 ccacggcgta acgcgcttgc tgcttggatg cccgaggcat agactgtaca aaaaaacagt
     9061 cataacaagc catgaaaacc gccactgcgc cgttaccacc gctgcgttcg gtcaaggttc
     9121 tggaccagtt gcgtgagcgc atacgctact tgcattacag tttacgaacc gaacaggctt
     9181 atgtcaatcc gatccattgc tgttgacaaa gggaatcagg ggatcttgaa gttcctattc
     9241 cgaagttcct attctctaga aagtatagga acttc
//
