LOCUS       sequence-209783-        5953 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing S. pasteurianus sgRNA with no 20bp target (Empty 
            control) Kanamycin marker for P. putida and P. fluorescens.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5953)
  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 5953)
  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-Kan
FEATURES             Location/Qualifiers
     source          1..5953
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(133..759)
                     /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
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     664..683
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(772..806)
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     promoter        807..862
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    843..861
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(938..954)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(986..1005)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(987..1005)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1015..1032)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1015..1031)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1024..1046)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(1740..2402)
                     /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      2403..3174
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             4654..5448
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4708..4727)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5318..5337
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    5632..5650
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5657..5675
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      5796..5839
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5890..5906
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      5907..5953
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctgttgtttg tcgcattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt
       61 tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagtt
      121 tttatataag ctttaagacc cactttcaca tttaagttgt ttttctaatc cgcatatgat
      181 caattcaagg ccgaataaga aggctggctc tgcaccttgg tgatcaaata attcgatagc
      241 ttgtcgtaat aatggcggca tactatcagt agtaggtgtt tccctttctt ctttagcgac
      301 ttgatgctct tgatcttcca atacgcaacc taaagtaaaa tgccccacag cgctgagtgc
      361 atataatgca ttctctagtg aaaaaccttg ttggcataaa aaggctaatt gattttcgag
      421 agtttcatac tgtttttctg taggccgtgt acctaaatgt acttttgctc catcgcgatg
      481 acttagtaaa gcacatctaa aacttttagc gttattacgt aaaaaatctt gccagctttc
      541 cccttctaaa gggcaaaagt gagtatggtg cctatctaac atctcaatgg ctaaggcgtc
      601 gagcaaagcc cgcttatttt ttacatgcca atacaatgta ggctgctcta cacctagctt
      661 ctgggcgagt ttacgggttg ttaaaccttc gattccgacc tcattaagca gctctaatgc
      721 gctgttaatc actttacttt tatctaatct agacatcatt aattcctaat tgctagcatt
      781 gtacctagga ctgagctagc cataaagttg acactctatc gttgatagag ttattttacc
      841 actccctatc agtgatagag aatctgcggc cgctgattag ctaagcagaa ggccatcctg
      901 acggatgcct ttttcgcttg ctagctgcag cccgggggat ccactagttc tagagcggcc
      961 gccaccgcgg tggagctcca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     1021 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     1081 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     1141 ccaacagttg cgcagcctga atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa
     1201 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccgt actgcgatga
     1261 gtggcagggc ggggcgtaat ttttttaagg cagttattgg tgcccttaaa cgcctggttg
     1321 ctacgcctga ataagtgata ataagcggat gaatggcaga aattcgaaag caaattcgac
     1381 ccggtcgtcg gttcagggca gggtcgttaa atagccgctt atgtctattg ctggtttacc
     1441 ggtttattga ctaccggaag cagtgtgacc gtgtgcttct caaatgcctg aggccagttt
     1501 gctcaggctc tccccgtgga ggtaataatt gacgatatga tcatttattc tgcctcccag
     1561 agcctgataa aaacggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg
     1621 tggcccggct ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcga
     1681 ggcggctaca gccgatagtc tggaacagcg cacttacggg ttgctgcgca acccaagtgc
     1741 taccggcgcg gcagcgtgac ccgtgtcggc ggctccaacg gctcgccatc gtccagaaaa
     1801 cacggctcat cgggcatcgg caggcgctgc tgcccgcgcc gttcccattc ctccgtttcg
     1861 gtcaaggctg gcaggtctgg ttccatgccc ggaatgccgg gctggctggg cggctcctcg
     1921 ccggggccgg tcggtagttg ctgctcgccc ggatacaggg tcgggatgcg gcgcaggtcg
     1981 ccatgcccca acagcgattc gtcctggtcg tcgtgatcaa ccaccacggc ggcactgaac
     2041 accgacaggc gcaactggtc gcggggctgg ccccacgcca cgcggtcatt gaccacgtag
     2101 gccgacacgg tgccggggcc gttgagcttc acgacggaga tccagcgctc ggccaccaag
     2161 tccttgactg cgtattggac cgtccgcaaa gaacgtccga tgagcttgga aagtgttttc
     2221 tggctgacca ccacggcgtt ctggtggccc atctgcgcca cgaggtgatg cagcagcatt
     2281 gccgccgtgg gtttcctcgc aataagcccg gcccacgcct catgcgcttt gcgttccgtt
     2341 tgcacccagt gaccgggctt gttcttggct tgaatgccga tttctctgga ctgcgtggcc
     2401 atgcttatct ccatgcggta ggggtgccgc acggttgcgg caccatgcgc aatcagctgc
     2461 aacttttcgg cagcgcgaca acaattatgc gttgcgtaaa agtggcagtc aattacagat
     2521 tttctttaac ctacgcaatg agctattgcg gggggtgccg caatgagctg ttgcgtaccc
     2581 ccctttttta agttgttgat ttttaagtct ttcgcatttc gccctatatc tagttctttg
     2641 gtgcccaaag aagggcaccc ctgcggggtt cccccacgcc ttcggcgcgg ctccccctcc
     2701 ggcaaaaagt ggcccctccg gggcttgttg atcgactgcg cggccttcgg ccttgcccaa
     2761 ggtggcgctg cccccttgga acccccgcac tcgccgccgt gaggctcggg gggcaggcgg
     2821 gcgggcttcg cccttcgact gcccccactc gcataggctt gggtcgttcc aggcgcgtca
     2881 aggccaagcc gctgcgcggt cgctgcgcga gccttgaccc gccttccact tggtgtccaa
     2941 ccggcaagcg aagcgcgcag gccgcaggcc ggaggctttt ccccagagaa aattaaaaaa
     3001 attgatgggg caaggccgca ggccgcgcag ttggagccgg tgggtatgtg gtcgaaggct
     3061 gggtagccgg tgggcaatcc ctgtggtcaa gctcgtgggc aggcgcagcc tgtccatcag
     3121 cttgtccagc agggttgtcc acgggccgag cgaagcgagc cagccggtgg ccgctcgcgg
     3181 ccatcgtcca catatccacg ggctggcaag ggagcgcagc gaccgcgcag ggcgaagccc
     3241 ggagagcaag cccgtagggc gccgcagccg ccgtaggcgg tcacgacttt gcgaagcaaa
     3301 gtctagtgag tatactcaag cattgagtgg cccgccggag gcaccgcctt gcgctgcccc
     3361 cgtcgagccg gttggacacc aaaagggagg ggcaggcatg gcggcatacg cgatcatgcg
     3421 atgcaagaag ctggcgaaaa tgggcaacgt ggcggccagt ctcaagcacg cctaccgcga
     3481 gcgcgagacg cccaacgctg acgccagcag gacgccagag aacgagcact gggcggccag
     3541 cagcaccgat gaagcgatgg gccgactgcg cgagttgctg ccagagaagc ggcgcaagga
     3601 cgctgtgttg gcggtcgagt acgtcatgac ggccagcccg gaatggtgga agtcggccag
     3661 ccaagaacag caggcggcgt tcttcgagaa ggcgcacaag tggctggcgg acaagtacgg
     3721 ggcggatcgc atcgtgacgg ccagcatcca ccgtgacgaa accagcccgc acatgaccgc
     3781 gttcgtggtg ccgctgacgc aggacggcag gctgtcggcc aaggagttca tcggcaacaa
     3841 agcgcagatg acccgcgacc agaccacgtt tgcggccgct gtggccgatc tagggctgca
     3901 acggggcatc gagggcagca aggcacgtca cacgcgcatt caggcgttct acgaggccct
     3961 ggagcggcca ccagtgggcc acgtcaccat cagcccgcaa gcggtcgagc cacgcgccta
     4021 tgcaccgcag ggattggccg aaaagctggg aatctcaaag cgcgttgaga cgccggaagc
     4081 cgtggccgac cggctgacaa aagcggttcg gcaggggtat gagcctgccc tacaggccgc
     4141 cgcaggagcg cgtgagatgc gcaagaaggc cgatcaagcc caagagacgg cccgagacct
     4201 tcgggagcgc ctgaagcccg ttctggacgc cctggggccg ttgaatcggg atatgcaggc
     4261 caaggccgcc gcgatcatca aggccgtggg cgaaaagctg ctgacggaac agcgggaagt
     4321 ccagcgccag aaacaggccc agcgccagca ggaacgcggg cgcgcacatt tccccgaaaa
     4381 gtgccacctg ggatgaatgt cagctactgg gctatctgga caagggaaaa cgcaagcgca
     4441 aagagaaagc aggtagcttg cagtgggctt acatggcgat agctagactg ggcggtttta
     4501 tggacagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc
     4561 tgcaaagtaa actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaaga
     4621 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     4681 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     4741 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     4801 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     4861 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     4921 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     4981 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     5041 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     5101 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     5161 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     5221 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     5281 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     5341 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     5401 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     5461 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     5521 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     5581 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc ccatgggcaa atccctatca
     5641 gtgatagaga ttgacatccc tatcagtgat agagatactg agcacacgtc ttcatgcagc
     5701 tgaagacttg tttttgtact cgaaagagcc tacaaagata aggctttatg ccgaattcaa
     5761 gcaccccatg ttttgacatg aggtgctttt ttttgataaa acgaaaggcc cagtctttcg
     5821 actgagcctt tcgttttata aacgggtctt gaggggtttt ttaagcttct gcagctggat
     5881 ccatggatat ctagaactag tggatccaaa taaaacgaaa ggctcagtcg aaagactggg
     5941 cctttcgttt tat
//
