LOCUS       sequence-436813-        4045 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA expression vector - pEM7 promoter with GFP in sgRNA site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4045)
  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 4045)
  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-GFP
FEATURES             Location/Qualifiers
     source          1..4045
                     /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        1012..1087
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1089..1132
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1225..1319
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1440..1459)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1451..2266
                     /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            2433..2541
                     /note="incP origin of transfer"
     CDS             complement(2552..3214)
                     /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      3215..3986
                     /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 tagcttatag aaggagatat acatatgatc atgggaattc ataaaggtga
      301 agaactgttc accggtgttg ttccgatcct ggttgaactg gatggtgatg ttaacggcca
      361 caaattctct gttcgtggtg aaggtgaagg tgatgcaacc aacggtaaac tgaccctgaa
      421 attcatctgc actaccggta aactgccggt tccatggccg actctggtga ctaccctgac
      481 ctatggtgtt cagtgttttt ctcgttaccc ggatcacatg aagcagcatg atttcttcaa
      541 atctgcaatg ccggaaggtt atgtacagga gcgcaccatt tctttcaaag acgatggcac
      601 ctacaaaacc cgtgcagagg ttaaatttga aggtgatact ctggtgaacc gtattgaact
      661 gaaaggcatt gatttcaaag aggacggcaa catcctgggc cacaaactgg aatataactt
      721 caactcccat aacgtttaca tcaccgcaga caaacagaag aacggtatca aagctaactt
      781 caaaattcgc cataacgttg aagacggtag cgtacagctg gcggaccact accagcagaa
      841 cactccgatc ggtgatggtc cggttctgct gccggataac cactacctgt ccacccagtc
      901 taaactgtcc aaagacccga acgaaaagcg cgaccacatg gtgctgctgg agttcgttac
      961 tgcagcaggt atcacgcacg gcatggatga actctacaaa taagggtctc tgttttagag
     1021 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     1081 tcggtgctat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt ataactaggt
     1141 acccggggat cctctagagt cgacctgcag gcatgcaagc ttgcggccgc gtcgtgactg
     1201 ggaaaaccct ggcgactagt cttggactcc tgttgataga tccagtaatg acctcagaac
     1261 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc
     1321 caggggtccc caataattac gatttaaatt tgtgtctcaa aatctctgat gttacattgc
     1381 acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac
     1441 aaggggtgtt atgagccata ttcagcgtga aacgagctgt agccgtccgc gtctgaacag
     1501 caacatggat gcggatctgt atggctataa atgggcgcgt gataacgtgg gtcagagcgg
     1561 cgcgaccatt tatcgtctgt atggcaaacc ggatgcgccg gaactgtttc tgaaacatgg
     1621 caaaggcagc gtggcgaacg atgtgaccga tgaaatggtg cgtctgaact ggctgaccga
     1681 atttatgccg ctgccgacca ttaaacattt tattcgcacc ccggatgatg cgtggctgct
     1741 gaccaccgcg attccgggca aaaccgcgtt tcaggtgctg gaagaatatc cggatagcgg
     1801 cgaaaacatt gtggatgcgc tggccgtgtt tctgcgtcgt ctgcatagca ttccggtgtg
     1861 caactgcccg tttaacagcg atcgtgtgtt tcgtctggcc caggcgcaga gccgtatgaa
     1921 caacggcctg gtggatgcga gcgattttga tgatgaacgt aacggctggc cggtggaaca
     1981 ggtgtggaaa gaaatgcata aactgctgcc gtttagcccg gatagcgtgg tgacccacgg
     2041 cgattttagc ctggataacc tgattttcga tgaaggcaaa ctgattggct gcattgatgt
     2101 gggccgtgtg ggcattgcgg atcgttatca ggatctggcc attctgtgga actgcctggg
     2161 cgaatttagc ccgagcctgc aaaaacgtct gtttcagaaa tatggcattg ataatccgga
     2221 tatgaacaaa ctgcaatttc atctgatgct ggatgaattt ttctaataat taattggacc
     2281 gcggtccgcg cgttgtcctt ttccgctgca taaccctgct tcggggtcat tatagcgatt
     2341 ttttcggtat atccatcctt tttcgcacga tatacaggat tttgccaaag ggttcgtgta
     2401 gactttcctt ggtgtatcca acggcgtcag ccgggcagga taggtgaagt aggcccaccc
     2461 gcgagcgggt gttccttctt cactgtccct tattcgcacc tggcggtgct caacgggaat
     2521 cctgctctgc gaggctggcc gtaggccggc cctaccggcg cggcagcgtt acccgtgtcg
     2581 gcggctccaa cggctcgcca tcgtccagaa aacacggctc atcgggcatc ggcaggcgct
     2641 gctgcccgcg ccgttcccat tcctccgttt cggtcaaggc tggcaggtct ggttccatgc
     2701 ccggaatgcc gggctggctg ggcggctcct cgccggggcc ggtcggtagt tgctgctcgc
     2761 ccggatacag ggtcgggatg cggcgcaggt cgccatgccc caacagcgat tcgtcctggt
     2821 cgtcgtgatc aaccaccacg gcggcactga acaccgacag gcgcaactgg tcgcggggct
     2881 ggccccacgc cacgcggtca ttgaccacgt aggccgacac ggtgccgggg ccgttgagct
     2941 tcacgacgga gatccagcgc tcggccacca agtccttgac tgcgtattgg accgtccgca
     3001 aagaacgtcc gatgagcttg gaaagtgtct tctggctgac caccacggcg ttctggtggc
     3061 ccatctgcgc cacgaggtga tgcagcagca ttgccgccgt gggtttcctc gcaataagcc
     3121 cggcccacgc ctcatgcgct ttgcgttccg tttgcaccca gtgaccgggc ttgttcttgg
     3181 cttgaatgcc gatttctctg gactgcgtgg ccatgcttat ctccatgcgg taggggtgcc
     3241 gcacggttgc ggcaccatgc gcaatcagct gcaacttttc ggcagcgcga caacaattat
     3301 gcgttgcgta aaagtggcag tcaattacag attttcttta acctacgcaa tgagctattg
     3361 cggggggtgc cgcaatgagc tgttgcgtac cccccttttt taagttgttg atttttaagt
     3421 ctttcgcatt tcgccctata tctagttctt tggtgcccaa agaagggcac ccctgcgggg
     3481 ttcccccacg ccttcggcgc ggctccccct ccggcaaaaa gtggcccctc cggggcttgt
     3541 tgatcgactg cgcggccttc ggccttgccc aaggtggcgc tgcccccttg gaacccccgc
     3601 actcgccgcc gtgaggctcg gggggcaggc gggcgggctt cgcccttcga ctgcccccac
     3661 tcgcataggc ttgggtcgtt ccaggcgcgt caaggccaag ccgctgcgcg gtcgctgcgc
     3721 gagccttgac ccgccttcca cttggtgtcc aaccggcaag cgaagcgcgc aggccgcagg
     3781 ccggaggctt ttccccagag aaaattaaaa aaattgatgg ggcaaggccg caggccgcgc
     3841 agttggagcc ggtgggtatg tggtcgaagg ctgggtagcc ggtgggcaat ccctgtggtc
     3901 aagctcgtgg gcaggcgcag cctgtccatc agcttgtcca gcagggttgt ccacgggccg
     3961 agcgaagcga gccagccggt ggccgctcgc ggccatcgtc cacatatcca cgggctggca
     4021 agggagcgca gcgaccgcgc agggc
//
