LOCUS       sequence-436812-        3294 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA expression vector - pEM7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3294)
  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 3294)
  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: pSpyA
FEATURES             Location/Qualifiers
     source          1..3294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(56..142)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        160..207
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     misc_RNA        261..336
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      338..381
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      474..568
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(689..708)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             700..1515
                     /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            1682..1790
                     /note="incP origin of transfer"
     CDS             complement(1801..2463)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      2464..3235
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
ORIGIN
        1 gaagcccgga gagcaagccc gtaggggggg cgcgcccagc tgtctagggc ggcggatttg
       61 tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg
      121 actgagcctt tcgttttatt tgatgccttt aattaacttg ttgacaatta atcatcggca
      181 tagtatatcg gcatagtata atacgacacc ctaggccgcg gccgcgcgaa ttcgagctcg
      241 gtctcatcac tagggtctct gttttagagc tagaaatagc aagttaaaat aaggctagtc
      301 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctata aaacgaaagg cccagtcttt
      361 cgactgagcc tttcgtttta taactaggta cccggggatc ctctagagtc gacctgcagg
      421 catgcaagct tgcggccgcg tcgtgactgg gaaaaccctg gcgactagtc ttggactcct
      481 gttgatagat ccagtaatga cctcagaact ccatctggat ttgttcagaa cgctcggttg
      541 ccgccgggcg ttttttattg gtgagaatcc aggggtcccc aataattacg atttaaattt
      601 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
      661 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcagcgtgaa
      721 acgagctgta gccgtccgcg tctgaacagc aacatggatg cggatctgta tggctataaa
      781 tgggcgcgtg ataacgtggg tcagagcggc gcgaccattt atcgtctgta tggcaaaccg
      841 gatgcgccgg aactgtttct gaaacatggc aaaggcagcg tggcgaacga tgtgaccgat
      901 gaaatggtgc gtctgaactg gctgaccgaa tttatgccgc tgccgaccat taaacatttt
      961 attcgcaccc cggatgatgc gtggctgctg accaccgcga ttccgggcaa aaccgcgttt
     1021 caggtgctgg aagaatatcc ggatagcggc gaaaacattg tggatgcgct ggccgtgttt
     1081 ctgcgtcgtc tgcatagcat tccggtgtgc aactgcccgt ttaacagcga tcgtgtgttt
     1141 cgtctggccc aggcgcagag ccgtatgaac aacggcctgg tggatgcgag cgattttgat
     1201 gatgaacgta acggctggcc ggtggaacag gtgtggaaag aaatgcataa actgctgccg
     1261 tttagcccgg atagcgtggt gacccacggc gattttagcc tggataacct gattttcgat
     1321 gaaggcaaac tgattggctg cattgatgtg ggccgtgtgg gcattgcgga tcgttatcag
     1381 gatctggcca ttctgtggaa ctgcctgggc gaatttagcc cgagcctgca aaaacgtctg
     1441 tttcagaaat atggcattga taatccggat atgaacaaac tgcaatttca tctgatgctg
     1501 gatgaatttt tctaataatt aattggaccg cggtccgcgc gttgtccttt tccgctgcat
     1561 aaccctgctt cggggtcatt atagcgattt tttcggtata tccatccttt ttcgcacgat
     1621 atacaggatt ttgccaaagg gttcgtgtag actttccttg gtgtatccaa cggcgtcagc
     1681 cgggcaggat aggtgaagta ggcccacccg cgagcgggtg ttccttcttc actgtccctt
     1741 attcgcacct ggcggtgctc aacgggaatc ctgctctgcg aggctggccg taggccggcc
     1801 ctaccggcgc ggcagcgtta cccgtgtcgg cggctccaac ggctcgccat cgtccagaaa
     1861 acacggctca tcgggcatcg gcaggcgctg ctgcccgcgc cgttcccatt cctccgtttc
     1921 ggtcaaggct ggcaggtctg gttccatgcc cggaatgccg ggctggctgg gcggctcctc
     1981 gccggggccg gtcggtagtt gctgctcgcc cggatacagg gtcgggatgc ggcgcaggtc
     2041 gccatgcccc aacagcgatt cgtcctggtc gtcgtgatca accaccacgg cggcactgaa
     2101 caccgacagg cgcaactggt cgcggggctg gccccacgcc acgcggtcat tgaccacgta
     2161 ggccgacacg gtgccggggc cgttgagctt cacgacggag atccagcgct cggccaccaa
     2221 gtccttgact gcgtattgga ccgtccgcaa agaacgtccg atgagcttgg aaagtgtctt
     2281 ctggctgacc accacggcgt tctggtggcc catctgcgcc acgaggtgat gcagcagcat
     2341 tgccgccgtg ggtttcctcg caataagccc ggcccacgcc tcatgcgctt tgcgttccgt
     2401 ttgcacccag tgaccgggct tgttcttggc ttgaatgccg atttctctgg actgcgtggc
     2461 catgcttatc tccatgcggt aggggtgccg cacggttgcg gcaccatgcg caatcagctg
     2521 caacttttcg gcagcgcgac aacaattatg cgttgcgtaa aagtggcagt caattacaga
     2581 ttttctttaa cctacgcaat gagctattgc ggggggtgcc gcaatgagct gttgcgtacc
     2641 cccctttttt aagttgttga tttttaagtc tttcgcattt cgccctatat ctagttcttt
     2701 ggtgcccaaa gaagggcacc cctgcggggt tcccccacgc cttcggcgcg gctccccctc
     2761 cggcaaaaag tggcccctcc ggggcttgtt gatcgactgc gcggccttcg gccttgccca
     2821 aggtggcgct gcccccttgg aacccccgca ctcgccgccg tgaggctcgg ggggcaggcg
     2881 ggcgggcttc gcccttcgac tgcccccact cgcataggct tgggtcgttc caggcgcgtc
     2941 aaggccaagc cgctgcgcgg tcgctgcgcg agccttgacc cgccttccac ttggtgtcca
     3001 accggcaagc gaagcgcgca ggccgcaggc cggaggcttt tccccagaga aaattaaaaa
     3061 aattgatggg gcaaggccgc aggccgcgca gttggagccg gtgggtatgt ggtcgaaggc
     3121 tgggtagccg gtgggcaatc cctgtggtca agctcgtggg caggcgcagc ctgtccatca
     3181 gcttgtccag cagggttgtc cacgggccga gcgaagcgag ccagccggtg gccgctcgcg
     3241 gccatcgtcc acatatccac gggctggcaa gggagcgcag cgaccgcgca gggc
//
