LOCUS       sequence-440437-        4033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA expression vector - pBG35 promoter with GFP in sgRNA site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4033)
  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 4033)
  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: pSpyB-GFP
FEATURES             Location/Qualifiers
     source          1..4033
                     /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"
     misc_RNA        1000..1075
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1077..1120
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1213..1307
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1428..1447)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1439..2254
                     /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            2421..2529
                     /note="incP origin of transfer"
     CDS             complement(2540..3202)
                     /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      3203..3974
                     /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 aattaattta tttgacatgc gtgatgttta
      181 gaattataat ttggggacct aggccgcggc cgcgcgaatt cgagctcggt ctcatcacta
      241 gcttatagaa ggagatatac atatgatcat gggaattcat aaaggtgaag aactgttcac
      301 cggtgttgtt ccgatcctgg ttgaactgga tggtgatgtt aacggccaca aattctctgt
      361 tcgtggtgaa ggtgaaggtg atgcaaccaa cggtaaactg accctgaaat tcatctgcac
      421 taccggtaaa ctgccggttc catggccgac tctggtgact accctgacct atggtgttca
      481 gtgtttttct cgttacccgg atcacatgaa gcagcatgat ttcttcaaat ctgcaatgcc
      541 ggaaggttat gtacaggagc gcaccatttc tttcaaagac gatggcacct acaaaacccg
      601 tgcagaggtt aaatttgaag gtgatactct ggtgaaccgt attgaactga aaggcattga
      661 tttcaaagag gacggcaaca tcctgggcca caaactggaa tataacttca actcccataa
      721 cgtttacatc accgcagaca aacagaagaa cggtatcaaa gctaacttca aaattcgcca
      781 taacgttgaa gacggtagcg tacagctggc ggaccactac cagcagaaca ctccgatcgg
      841 tgatggtccg gttctgctgc cggataacca ctacctgtcc acccagtcta aactgtccaa
      901 agacccgaac gaaaagcgcg accacatggt gctgctggag ttcgttactg cagcaggtat
      961 cacgcacggc atggatgaac tctacaaata agggtctctg ttttagagct agaaatagca
     1021 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgctataa
     1081 aacgaaaggc ccagtctttc gactgagcct ttcgttttat aactaggtac ccggggatcc
     1141 tctagagtcg acctgcaggc atgcaagctt gcggccgcgt cgtgactggg aaaaccctgg
     1201 cgactagtct tggactcctg ttgatagatc cagtaatgac ctcagaactc catctggatt
     1261 tgttcagaac gctcggttgc cgccgggcgt tttttattgg tgagaatcca ggggtcccca
     1321 ataattacga tttaaatttg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa
     1381 tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     1441 gagccatatt cagcgtgaaa cgagctgtag ccgtccgcgt ctgaacagca acatggatgc
     1501 ggatctgtat ggctataaat gggcgcgtga taacgtgggt cagagcggcg cgaccattta
     1561 tcgtctgtat ggcaaaccgg atgcgccgga actgtttctg aaacatggca aaggcagcgt
     1621 ggcgaacgat gtgaccgatg aaatggtgcg tctgaactgg ctgaccgaat ttatgccgct
     1681 gccgaccatt aaacatttta ttcgcacccc ggatgatgcg tggctgctga ccaccgcgat
     1741 tccgggcaaa accgcgtttc aggtgctgga agaatatccg gatagcggcg aaaacattgt
     1801 ggatgcgctg gccgtgtttc tgcgtcgtct gcatagcatt ccggtgtgca actgcccgtt
     1861 taacagcgat cgtgtgtttc gtctggccca ggcgcagagc cgtatgaaca acggcctggt
     1921 ggatgcgagc gattttgatg atgaacgtaa cggctggccg gtggaacagg tgtggaaaga
     1981 aatgcataaa ctgctgccgt ttagcccgga tagcgtggtg acccacggcg attttagcct
     2041 ggataacctg attttcgatg aaggcaaact gattggctgc attgatgtgg gccgtgtggg
     2101 cattgcggat cgttatcagg atctggccat tctgtggaac tgcctgggcg aatttagccc
     2161 gagcctgcaa aaacgtctgt ttcagaaata tggcattgat aatccggata tgaacaaact
     2221 gcaatttcat ctgatgctgg atgaattttt ctaataatta attggaccgc ggtccgcgcg
     2281 ttgtcctttt ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatat
     2341 ccatcctttt tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg
     2401 tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgt
     2461 tccttcttca ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga
     2521 ggctggccgt aggccggccc taccggcgcg gcagcgttac ccgtgtcggc ggctccaacg
     2581 gctcgccatc gtccagaaaa cacggctcat cgggcatcgg caggcgctgc tgcccgcgcc
     2641 gttcccattc ctccgtttcg gtcaaggctg gcaggtctgg ttccatgccc ggaatgccgg
     2701 gctggctggg cggctcctcg ccggggccgg tcggtagttg ctgctcgccc ggatacaggg
     2761 tcgggatgcg gcgcaggtcg ccatgcccca acagcgattc gtcctggtcg tcgtgatcaa
     2821 ccaccacggc ggcactgaac accgacaggc gcaactggtc gcggggctgg ccccacgcca
     2881 cgcggtcatt gaccacgtag gccgacacgg tgccggggcc gttgagcttc acgacggaga
     2941 tccagcgctc ggccaccaag tccttgactg cgtattggac cgtccgcaaa gaacgtccga
     3001 tgagcttgga aagtgtcttc tggctgacca ccacggcgtt ctggtggccc atctgcgcca
     3061 cgaggtgatg cagcagcatt gccgccgtgg gtttcctcgc aataagcccg gcccacgcct
     3121 catgcgcttt gcgttccgtt tgcacccagt gaccgggctt gttcttggct tgaatgccga
     3181 tttctctgga ctgcgtggcc atgcttatct ccatgcggta ggggtgccgc acggttgcgg
     3241 caccatgcgc aatcagctgc aacttttcgg cagcgcgaca acaattatgc gttgcgtaaa
     3301 agtggcagtc aattacagat tttctttaac ctacgcaatg agctattgcg gggggtgccg
     3361 caatgagctg ttgcgtaccc ccctttttta agttgttgat ttttaagtct ttcgcatttc
     3421 gccctatatc tagttctttg gtgcccaaag aagggcaccc ctgcggggtt cccccacgcc
     3481 ttcggcgcgg ctccccctcc ggcaaaaagt ggcccctccg gggcttgttg atcgactgcg
     3541 cggccttcgg ccttgcccaa ggtggcgctg cccccttgga acccccgcac tcgccgccgt
     3601 gaggctcggg gggcaggcgg gcgggcttcg cccttcgact gcccccactc gcataggctt
     3661 gggtcgttcc aggcgcgtca aggccaagcc gctgcgcggt cgctgcgcga gccttgaccc
     3721 gccttccact tggtgtccaa ccggcaagcg aagcgcgcag gccgcaggcc ggaggctttt
     3781 ccccagagaa aattaaaaaa attgatgggg caaggccgca ggccgcgcag ttggagccgg
     3841 tgggtatgtg gtcgaaggct gggtagccgg tgggcaatcc ctgtggtcaa gctcgtgggc
     3901 aggcgcagcc tgtccatcag cttgtccagc agggttgtcc acgggccgag cgaagcgagc
     3961 cagccggtgg ccgctcgcgg ccatcgtcca catatccacg ggctggcaag ggagcgcagc
     4021 gaccgcgcag ggc
//
