LOCUS       sequence-210662-        5599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing S. pasteurianus sgRNA with no 20bp target (Empty 
            control) Gentamycin marker for P. aeruginosa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5599)
  AUTHORS   Tan SZ, Reisch CR, Prather KLJ
  TITLE     A Robust CRISPR Interference Gene Repression System in Pseudomonas.
  JOURNAL   J Bacteriol. 2018 Mar 12;200(7). pii: JB.00575-17. doi: 
            10.1128/JB.00575-17. Print 2018 Apr 1.
  PUBMED    29311279
REFERENCE   2  (bases 1 to 5599)
  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: "J 
            Bacteriol."; date: "2018-03-12"; volume: "200(7). pii: JB.00575-17. 
            doi"; pages: " 10.1128/JB.00575-17. Print 2018 Apr 1"
COMMENT     Sequence Label: pBx-Spas-sgRNA-Gm
FEATURES             Location/Qualifiers
     source          1..5599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..534
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     protein_bind    731..749
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    756..774
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     989..1005
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     989..1005
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      1006..1052
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1185..1811)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAI*MLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     1716..1735
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(1824..1858)
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     promoter        1859..1914
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    1895..1913
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(1932..1951)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1933..1951)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1961..1978)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1961..1977)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1970..1992)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(2686..3348)
                     /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      3349..4120
                     /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 atgttacgca gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaggt
       61 ggctcaagta tgggcatcat tcgcacatgt aggctcggcc ctgaccaagt caaatccatg
      121 cgggctgctc ttgatctttt cggtcgtgag ttcggagacg tagccaccta ctcccaacat
      181 cagccggact ccgattacct cgggaacttg ctccgtagta agacattcat cgcgcttgct
      241 gccttcgacc aagaagcggt tgttggcgct ctcgcggctt acgttctgcc caagtttgag
      301 cagccgcgta gtgagatcta tatctatgat ctcgcagtct ccggagagca ccggaggcag
      361 ggcattgcca ccgcgctcat caatctcctc aagcatgagg ccaacgcgct tggtgcttat
      421 gtgatctacg tgcaagcaga ttacggtgac gatcccgcag tggctctcta tacaaagttg
      481 ggcatacggg aagaagtgat gcactttgat atcgacccaa gtaccgccac ctaacgagtt
      541 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
      601 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
      661 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
      721 catgggcaaa tccctatcag tgatagagat tgacatccct atcagtgata gagatactga
      781 gcacacgtct tcatgcagct gaagacttgt ttttgtactc gaaagagcct acaaagataa
      841 ggctttatgc cgaattcaag caccccatgt tttgacatga ggtgcttttt tttgataaaa
      901 cgaaaggccc agtctttcta ctgagccttt cgttttataa acgggtcttg aggggttttt
      961 taagcttctg cagctggatc catggatatc tagaactagt ggatccaaat aaaacgaaag
     1021 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcgcatta tacgcaaggc
     1081 gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat
     1141 gtcggcagaa tgcttaatga attacaacag tttttatata agctttaaga cccactttca
     1201 catttaagtt gtttttctaa tccgcatatg atcaattcaa ggccgaataa gaaggctggc
     1261 tctgcacctt ggtgatcaaa taattcgata gcttgtcgta ataatggcgg catactatca
     1321 gtagtaggtg tttccctttc ttctttagcg acttgatgct cttgatcttc caatacgcaa
     1381 cctaaagtaa aatgccccac agcgctgagt gcatataatg cattctctag tgaaaaacct
     1441 tgttggcata aaaaggctaa ttgattttcg agagtttcat actgtttttc tgtaggccgt
     1501 gtacctaaat gtacttttgc tccatcgcga tgacttagta aagcacatct aaaactttta
     1561 gcgttattac gtaaaaaatc ttgccagctt tccccttcta aagggcaaaa gtgagtatgg
     1621 tgcctatcta acatctaaat ggctaaggcg tcgagcaaag cccgcttatt ttttacatgc
     1681 caatacaatg taggctgctc tacacctagc ttctgggcga gtttacgggt tgttaaacct
     1741 tcgattccga cctcattaag cagctctaat gcgctgttaa tcactttact tttatctaat
     1801 ctagacatca ttaattccta attgctagca ttgtacctag gactgagcta gccataaagt
     1861 tgacactcta tcgttgatag agttatttta ccactcccta tcagtgatag agaatctcga
     1921 gctccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     1981 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     2041 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     2101 gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     2161 ttgttaaatc agctcatttt ttaaccaata ggccgtactg cgatgagtgg cagggcgggg
     2221 cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc tggttgctac gcctgaataa
     2281 gtgataataa gcggatgaat ggcagaaatt cgaaagcaaa ttcgacccgg tcgtcggttc
     2341 agggcagggt cgttaaatag ccgcttatgt ctattgctgg tttaccggtt tattgactac
     2401 cggaagcagt gtgaccgtgt gcttctcaaa tgcctgaggc cagtttgctc aggctctccc
     2461 cgtggaggta ataattgacg atatgatcat ttattctgcc tcccagagcc tgataaaaac
     2521 ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat
     2581 gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgaggcg gctacagccg
     2641 atagtctgga acagcgcact tacgggttgc tgcgcaaccc aagtgctacc ggcgcggcag
     2701 cgtgacccgt gtcggcggct ccaacggctc gccatcgtcc agaaaacacg gctcatcggg
     2761 catcggcagg cgctgctgcc cgcgccgttc ccattcctcc gtttcggtca aggctggcag
     2821 gtctggttcc atgcccggaa tgccgggctg gctgggcggc tcctcgccgg ggccggtcgg
     2881 tagttgctgc tcgcccggat acagggtcgg gatgcggcgc aggtcgccat gccccaacag
     2941 cgattcgtcc tggtcgtcgt gatcaaccac cacggcggca ctgaacaccg acaggcgcaa
     3001 ctggtcgcgg ggctggcccc acgccacgcg gtcattgacc acgtaggccg acacggtgcc
     3061 ggggccgttg agcttcacga cggagatcca gcgctcggcc accaagtcct tgactgcgta
     3121 ttggaccgtc cgcaaagaac gtccgatgag cttggaaagt gttttctggc tgaccaccac
     3181 ggcgttctgg tggcccatct gcgccacgag gtgatgcagc agcattgccg ccgtgggttt
     3241 cctcgcaata agcccggccc acgcctcatg cgctttgcgt tccgtttgca cccagtgacc
     3301 gggcttgttc ttggcttgaa tgccgatttc tctggactgc gtggccatgc ttatctccat
     3361 gcggtagggg tgccgcacgg ttgcggcacc atgcgcaatc agctgcaact tttcggcagc
     3421 gcgacaacaa ttatgcgttg cgtaaaagtg gcagtcaatt acagattttc tttaacctac
     3481 gcaatgagct attgcggggg gtgccgcaat gagctgttgc gtacccccct tttttaagtt
     3541 gttgattttt aagtctttcg catttcgccc tatatctagt tctttggtgc ccaaagaagg
     3601 gcacccctgc ggggttcccc cacgccttcg gcgcggctcc ccctccggca aaaagtggcc
     3661 cctccggggc ttgttgatcg actgcgcggc cttcggcctt gcccaaggtg gcgctgcccc
     3721 cttggaaccc ccgcactcgc cgccgtgagg ctcggggggc aggcgggcgg gcttcgccct
     3781 tcgactgccc ccactcgcat aggcttgggt cgttccaggc gcgtcaaggc caagccgctg
     3841 cgcggtcgct gcgcgagcct tgacccgcct tccacttggt gtccaaccgg caagcgaagc
     3901 gcgcaggccg caggccggag gcttttcccc agagaaaatt aaaaaaattg atggggcaag
     3961 gccgcaggcc gcgcagttgg agccggtggg tatgtggtcg aaggctgggt agccggtggg
     4021 caatccctgt ggtcaagctc gtgggcaggc gcagcctgtc catcagcttg tccagcaggg
     4081 ttgtccacgg gccgagcgaa gcgagccagc cggtggccgc tcgcggccat cgtccacata
     4141 tccacgggct ggcaagggag cgcagcgacc gcgcagggcg aagcccggag agcaagcccg
     4201 tagggcgccg cagccgccgt aggcggtcac gactttgcga agcaaagtct agtgagtata
     4261 ctcaagcatt gagtggcccg ccggaggcac cgccttgcgc tgcccccgtc gagccggttg
     4321 gacaccaaaa gggaggggca ggcatggcgg catacgcgat catgcgatgc aagaagctgg
     4381 cgaaaatggg caacgtggcg gccagtctca agcacgccta ccgcgagcgc gagacgccca
     4441 acgctgacgc cagcaggacg ccagagaacg agcactgggc ggccagcagc accgatgaag
     4501 cgatgggccg actgcgcgag ttgctgccag agaagcggcg caaggacgct gtgttggcgg
     4561 tcgagtacgt catgacggcc agcccggaat ggtggaagtc ggccagccaa gaacagcagg
     4621 cggcgttctt cgagaaggcg cacaagtggc tggcggacaa gtacggggcg gatcgcatcg
     4681 tgacggccag catccaccgt gacgaaacca gcccgcacat gaccgcgttc gtggtgccgc
     4741 tgacgcagga cggcaggctg tcggccaagg agttcatcgg caacaaagcg cagatgaccc
     4801 gcgaccagac cacgtttgcg gccgctgtgg ccgatctagg gctgcaacgg ggcatcgagg
     4861 gcagcaaggc acgtcacacg cgcattcagg cgttctacga ggccctggag cggccaccag
     4921 tgggccacgt caccatcagc ccgcaagcgg tcgagccacg cgcctatgca ccgcagggat
     4981 tggccgaaaa gctgggaatc tcaaagcgcg ttgagacgcc ggaagccgtg gccgaccggc
     5041 tgacaaaagc ggttcggcag gggtatgagc ctgccctaca ggccgccgca ggagcgcgtg
     5101 agatgcgcaa gaaggccgat caagcccaag agacggcccg agaccttcgg gagcgcctga
     5161 agcccgttct ggacgccctg gggccgttga atcgggatat gcaggcaaag gccgccgcga
     5221 tcatcaaggc cgtgggcgaa aagctgctga cggaacagcg ggaagtccag cgccagaaac
     5281 aggcccagcg ccagcaggaa cgcgggcgcg cacatttccc cgaaaagtgc cacctgggat
     5341 gaatgtcagc tactgggcta tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt
     5401 agcttgcagt gggcttacat ggcgatagct agactgggcg gttttatgga cagcaagcga
     5461 accggaattg ccagctgggg cgccctctgg taaggttggg aagccctgca aagtaaactg
     5521 gatggctttc ttgccgccaa ggatctgatg gcgcagggga tcaagatctg atcaagagac
     5581 aggatgagga tcgtttcgc
//
