LOCUS       sequence-440436-        3270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA expression vector - promoterless backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3270)
  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 3270)
  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: pSpy
FEATURES             Location/Qualifiers
     source          1..3270
                     /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"
     primer_bind     157..179
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_RNA        237..312
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      314..357
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      450..544
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(665..684)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             676..1491
                     /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            1658..1766
                     /note="incP origin of transfer"
     CDS             complement(1777..2439)
                     /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      2440..3211
                     /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 aattaaagcg gataacaatt tcacacagga
      181 ggccgcctag gccgcggccg cgcgaattcg agctcggtct catcactagg gtctctgttt
      241 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
      301 ccgagtcggt gctataaaac gaaaggccca gtctttcgac tgagcctttc gttttataac
      361 taggtacccg gggatcctct agagtcgacc tgcaggcatg caagcttgcg gccgcgtcgt
      421 gactgggaaa accctggcga ctagtcttgg actcctgttg atagatccag taatgacctc
      481 agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga
      541 gaatccaggg gtccccaata attacgattt aaatttgtgt ctcaaaatct ctgatgttac
      601 attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt
      661 aatacaaggg gtgttatgag ccatattcag cgtgaaacga gctgtagccg tccgcgtctg
      721 aacagcaaca tggatgcgga tctgtatggc tataaatggg cgcgtgataa cgtgggtcag
      781 agcggcgcga ccatttatcg tctgtatggc aaaccggatg cgccggaact gtttctgaaa
      841 catggcaaag gcagcgtggc gaacgatgtg accgatgaaa tggtgcgtct gaactggctg
      901 accgaattta tgccgctgcc gaccattaaa cattttattc gcaccccgga tgatgcgtgg
      961 ctgctgacca ccgcgattcc gggcaaaacc gcgtttcagg tgctggaaga atatccggat
     1021 agcggcgaaa acattgtgga tgcgctggcc gtgtttctgc gtcgtctgca tagcattccg
     1081 gtgtgcaact gcccgtttaa cagcgatcgt gtgtttcgtc tggcccaggc gcagagccgt
     1141 atgaacaacg gcctggtgga tgcgagcgat tttgatgatg aacgtaacgg ctggccggtg
     1201 gaacaggtgt ggaaagaaat gcataaactg ctgccgttta gcccggatag cgtggtgacc
     1261 cacggcgatt ttagcctgga taacctgatt ttcgatgaag gcaaactgat tggctgcatt
     1321 gatgtgggcc gtgtgggcat tgcggatcgt tatcaggatc tggccattct gtggaactgc
     1381 ctgggcgaat ttagcccgag cctgcaaaaa cgtctgtttc agaaatatgg cattgataat
     1441 ccggatatga acaaactgca atttcatctg atgctggatg aatttttcta ataattaatt
     1501 ggaccgcggt ccgcgcgttg tccttttccg ctgcataacc ctgcttcggg gtcattatag
     1561 cgattttttc ggtatatcca tcctttttcg cacgatatac aggattttgc caaagggttc
     1621 gtgtagactt tccttggtgt atccaacggc gtcagccggg caggataggt gaagtaggcc
     1681 cacccgcgag cgggtgttcc ttcttcactg tcccttattc gcacctggcg gtgctcaacg
     1741 ggaatcctgc tctgcgaggc tggccgtagg ccggccctac cggcgcggca gcgttacccg
     1801 tgtcggcggc tccaacggct cgccatcgtc cagaaaacac ggctcatcgg gcatcggcag
     1861 gcgctgctgc ccgcgccgtt cccattcctc cgtttcggtc aaggctggca ggtctggttc
     1921 catgcccgga atgccgggct ggctgggcgg ctcctcgccg gggccggtcg gtagttgctg
     1981 ctcgcccgga tacagggtcg ggatgcggcg caggtcgcca tgccccaaca gcgattcgtc
     2041 ctggtcgtcg tgatcaacca ccacggcggc actgaacacc gacaggcgca actggtcgcg
     2101 gggctggccc cacgccacgc ggtcattgac cacgtaggcc gacacggtgc cggggccgtt
     2161 gagcttcacg acggagatcc agcgctcggc caccaagtcc ttgactgcgt attggaccgt
     2221 ccgcaaagaa cgtccgatga gcttggaaag tgtcttctgg ctgaccacca cggcgttctg
     2281 gtggcccatc tgcgccacga ggtgatgcag cagcattgcc gccgtgggtt tcctcgcaat
     2341 aagcccggcc cacgcctcat gcgctttgcg ttccgtttgc acccagtgac cgggcttgtt
     2401 cttggcttga atgccgattt ctctggactg cgtggccatg cttatctcca tgcggtaggg
     2461 gtgccgcacg gttgcggcac catgcgcaat cagctgcaac ttttcggcag cgcgacaaca
     2521 attatgcgtt gcgtaaaagt ggcagtcaat tacagatttt ctttaaccta cgcaatgagc
     2581 tattgcgggg ggtgccgcaa tgagctgttg cgtacccccc ttttttaagt tgttgatttt
     2641 taagtctttc gcatttcgcc ctatatctag ttctttggtg cccaaagaag ggcacccctg
     2701 cggggttccc ccacgccttc ggcgcggctc cccctccggc aaaaagtggc ccctccgggg
     2761 cttgttgatc gactgcgcgg ccttcggcct tgcccaaggt ggcgctgccc ccttggaacc
     2821 cccgcactcg ccgccgtgag gctcgggggg caggcgggcg ggcttcgccc ttcgactgcc
     2881 cccactcgca taggcttggg tcgttccagg cgcgtcaagg ccaagccgct gcgcggtcgc
     2941 tgcgcgagcc ttgacccgcc ttccacttgg tgtccaaccg gcaagcgaag cgcgcaggcc
     3001 gcaggccgga ggcttttccc cagagaaaat taaaaaaatt gatggggcaa ggccgcaggc
     3061 cgcgcagttg gagccggtgg gtatgtggtc gaaggctggg tagccggtgg gcaatccctg
     3121 tggtcaagct cgtgggcagg cgcagcctgt ccatcagctt gtccagcagg gttgtccacg
     3181 ggccgagcga agcgagccag ccggtggccg ctcgcggcca tcgtccacat atccacgggc
     3241 tggcaaggga gcgcagcgac cgcgcagggc
//
