LOCUS       sequence-440441-        4020 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA expression vector - pBG28 promoter with GFP in sgRNA site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4020)
  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 4020)
  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: pSpyD-GFP
FEATURES             Location/Qualifiers
     source          1..4020
                     /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        987..1062
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1064..1107
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1200..1294
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1415..1434)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1426..2241
                     /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            2408..2516
                     /note="incP origin of transfer"
     CDS             complement(2527..3189)
                     /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      3190..3961
                     /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 aattaactag gttgacatgg atataatgta
      181 tgtacctagg ccgcggccgc gcgaattcga gctcggtctc atcactagct tatagaagga
      241 gatatacata tgatcatggg aattcataaa ggtgaagaac tgttcaccgg tgttgttccg
      301 atcctggttg aactggatgg tgatgttaac ggccacaaat tctctgttcg tggtgaaggt
      361 gaaggtgatg caaccaacgg taaactgacc ctgaaattca tctgcactac cggtaaactg
      421 ccggttccat ggccgactct ggtgactacc ctgacctatg gtgttcagtg tttttctcgt
      481 tacccggatc acatgaagca gcatgatttc ttcaaatctg caatgccgga aggttatgta
      541 caggagcgca ccatttcttt caaagacgat ggcacctaca aaacccgtgc agaggttaaa
      601 tttgaaggtg atactctggt gaaccgtatt gaactgaaag gcattgattt caaagaggac
      661 ggcaacatcc tgggccacaa actggaatat aacttcaact cccataacgt ttacatcacc
      721 gcagacaaac agaagaacgg tatcaaagct aacttcaaaa ttcgccataa cgttgaagac
      781 ggtagcgtac agctggcgga ccactaccag cagaacactc cgatcggtga tggtccggtt
      841 ctgctgccgg ataaccacta cctgtccacc cagtctaaac tgtccaaaga cccgaacgaa
      901 aagcgcgacc acatggtgct gctggagttc gttactgcag caggtatcac gcacggcatg
      961 gatgaactct acaaataagg gtctctgttt tagagctaga aatagcaagt taaaataagg
     1021 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctataaaac gaaaggccca
     1081 gtctttcgac tgagcctttc gttttataac taggtacccg gggatcctct agagtcgacc
     1141 tgcaggcatg caagcttgcg gccgcgtcgt gactgggaaa accctggcga ctagtcttgg
     1201 actcctgttg atagatccag taatgacctc agaactccat ctggatttgt tcagaacgct
     1261 cggttgccgc cgggcgtttt ttattggtga gaatccaggg gtccccaata attacgattt
     1321 aaatttgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga
     1381 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcag
     1441 cgtgaaacga gctgtagccg tccgcgtctg aacagcaaca tggatgcgga tctgtatggc
     1501 tataaatggg cgcgtgataa cgtgggtcag agcggcgcga ccatttatcg tctgtatggc
     1561 aaaccggatg cgccggaact gtttctgaaa catggcaaag gcagcgtggc gaacgatgtg
     1621 accgatgaaa tggtgcgtct gaactggctg accgaattta tgccgctgcc gaccattaaa
     1681 cattttattc gcaccccgga tgatgcgtgg ctgctgacca ccgcgattcc gggcaaaacc
     1741 gcgtttcagg tgctggaaga atatccggat agcggcgaaa acattgtgga tgcgctggcc
     1801 gtgtttctgc gtcgtctgca tagcattccg gtgtgcaact gcccgtttaa cagcgatcgt
     1861 gtgtttcgtc tggcccaggc gcagagccgt atgaacaacg gcctggtgga tgcgagcgat
     1921 tttgatgatg aacgtaacgg ctggccggtg gaacaggtgt ggaaagaaat gcataaactg
     1981 ctgccgttta gcccggatag cgtggtgacc cacggcgatt ttagcctgga taacctgatt
     2041 ttcgatgaag gcaaactgat tggctgcatt gatgtgggcc gtgtgggcat tgcggatcgt
     2101 tatcaggatc tggccattct gtggaactgc ctgggcgaat ttagcccgag cctgcaaaaa
     2161 cgtctgtttc agaaatatgg cattgataat ccggatatga acaaactgca atttcatctg
     2221 atgctggatg aatttttcta ataattaatt ggaccgcggt ccgcgcgttg tccttttccg
     2281 ctgcataacc ctgcttcggg gtcattatag cgattttttc ggtatatcca tcctttttcg
     2341 cacgatatac aggattttgc caaagggttc gtgtagactt tccttggtgt atccaacggc
     2401 gtcagccggg caggataggt gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg
     2461 tcccttattc gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc tggccgtagg
     2521 ccggccctac cggcgcggca gcgttacccg tgtcggcggc tccaacggct cgccatcgtc
     2581 cagaaaacac ggctcatcgg gcatcggcag gcgctgctgc ccgcgccgtt cccattcctc
     2641 cgtttcggtc aaggctggca ggtctggttc catgcccgga atgccgggct ggctgggcgg
     2701 ctcctcgccg gggccggtcg gtagttgctg ctcgcccgga tacagggtcg ggatgcggcg
     2761 caggtcgcca tgccccaaca gcgattcgtc ctggtcgtcg tgatcaacca ccacggcggc
     2821 actgaacacc gacaggcgca actggtcgcg gggctggccc cacgccacgc ggtcattgac
     2881 cacgtaggcc gacacggtgc cggggccgtt gagcttcacg acggagatcc agcgctcggc
     2941 caccaagtcc ttgactgcgt attggaccgt ccgcaaagaa cgtccgatga gcttggaaag
     3001 tgtcttctgg ctgaccacca cggcgttctg gtggcccatc tgcgccacga ggtgatgcag
     3061 cagcattgcc gccgtgggtt tcctcgcaat aagcccggcc cacgcctcat gcgctttgcg
     3121 ttccgtttgc acccagtgac cgggcttgtt cttggcttga atgccgattt ctctggactg
     3181 cgtggccatg cttatctcca tgcggtaggg gtgccgcacg gttgcggcac catgcgcaat
     3241 cagctgcaac ttttcggcag cgcgacaaca attatgcgtt gcgtaaaagt ggcagtcaat
     3301 tacagatttt ctttaaccta cgcaatgagc tattgcgggg ggtgccgcaa tgagctgttg
     3361 cgtacccccc ttttttaagt tgttgatttt taagtctttc gcatttcgcc ctatatctag
     3421 ttctttggtg cccaaagaag ggcacccctg cggggttccc ccacgccttc ggcgcggctc
     3481 cccctccggc aaaaagtggc ccctccgggg cttgttgatc gactgcgcgg ccttcggcct
     3541 tgcccaaggt ggcgctgccc ccttggaacc cccgcactcg ccgccgtgag gctcgggggg
     3601 caggcgggcg ggcttcgccc ttcgactgcc cccactcgca taggcttggg tcgttccagg
     3661 cgcgtcaagg ccaagccgct gcgcggtcgc tgcgcgagcc ttgacccgcc ttccacttgg
     3721 tgtccaaccg gcaagcgaag cgcgcaggcc gcaggccgga ggcttttccc cagagaaaat
     3781 taaaaaaatt gatggggcaa ggccgcaggc cgcgcagttg gagccggtgg gtatgtggtc
     3841 gaaggctggg tagccggtgg gcaatccctg tggtcaagct cgtgggcagg cgcagcctgt
     3901 ccatcagctt gtccagcagg gttgtccacg ggccgagcga agcgagccag ccggtggccg
     3961 ctcgcggcca tcgtccacat atccacgggc tggcaaggga gcgcagcgac cgcgcagggc
//
