LOCUS       sequence-364506-        8670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli - B. subtilis nicking vector pProUSER13G2F . Ap-BBR1; 
            rsbP-insertion-site; Ery; tetR-Ptet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8670)
  AUTHORS   Falkenberg KB, Mol V, de la Maza Larrea AS, Pogrebnyakov I, Norholm 
            MHH, Nielsen AT, Jensen SI
  TITLE     The ProUSER2.0 Toolbox: Genetic Parts and Highly Customizable 
            Plasmids for Synthetic Biology in Bacillus subtilis.
  JOURNAL   ACS Synth Biol. 2021 Dec 17;10(12):3278-3289. doi: 
            10.1021/acssynbio.1c00130. Epub 2021 Nov 18.
  PUBMED    34793671
REFERENCE   2  (bases 1 to 8670)
  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"; doi: 
            "10.1021/acssynbio.1c00130"; journalName: "ACS Synth Biol"; date: 
            "2021-12-17- 17"; volume: "10"; issue: "12"; pages: "3278-3289"
COMMENT     Sequence Label: pProUSER13G2F
FEATURES             Location/Qualifiers
     source          1..8670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(54..140)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     155..177
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             complement(191..817)
                     /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     722..741
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    924..942
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     terminator      2852..2946
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        5132..5223
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5224..5292,5293..6084)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     terminator      6088..6127
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            6274..6382
                     /note="incP origin of transfer"
     CDS             complement(6393..7055)
                     /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      7056..7827
                     /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 ggattttttt gttaccagct tgtacaggcg cgcccagctg tctagggcgg cggatttgtc
       61 ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac
      121 tgagcctttc gttttatttg atgcctttaa ttaaagcgga taacaatttc acacaggagg
      181 ccgatcgcgt ttaagaccca ctttcacatt taagttgttt ttctaatccg catatgatca
      241 attcaaggcc gaataagaag gctggctctg caccttggtg atcaaataat tcgatagctt
      301 gtcgtaataa tggcggcata ctatcagtag taggtgtttc cctttcttct ttagcgactt
      361 gatgctcttg atcttccaat acgcaaccta aagtaaaatg ccccacagcg ctgagtgcat
      421 ataatgcatt ctctagtgaa aaaccttgtt ggcataaaaa ggctaattga ttttcgagag
      481 tttcatactg tttttctgta ggccgtgtac ctaaatgtac ttttgctcca tcgcgatgac
      541 ttagtaaagc acatctaaaa cttttagcgt tattacgtaa aaaatcttgc cagctttccc
      601 cttctaaagg gcaaaagtga gtatggtgcc tatctaacat ctcaatggct aaggcgtcga
      661 gcaaagcccg cttatttttt acatgccaat acaatgtagg ctgctctaca cctagcttct
      721 gggcgagttt acgggttgtt aaaccttcga ttccgacctc attaagcagc tctaatgcgc
      781 tgttaatcac tttactttta tctaatctag acatcattaa ttcctccttt ttgttgacat
      841 tatatcattg atagagttat ttgtcaaact agatttttat ttcgatgccc tggacttcat
      901 gaaaaactaa aaaaaatatt gacactctat cattgataga gtataattaa aataagcttg
      961 atcgtagcgt taacagatct gagctcctgc agtaagctga ggcgatcgca acgtcgtgac
     1021 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     1081 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     1141 ggcgaatggc gctttgcctg gtttccggca ccagaagcgg tgccggaaag ctggctggag
     1201 tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa actggcagat gcacggttac
     1261 gatgcgccca tctacaccaa cgtgacctat cccattacgg tcaatccgcc gtttgttccc
     1321 acggagaatc cgacgggttg ttactcgctc acatttaatg ttgatgaaag ctggctacag
     1381 gaaggccaga cgcgaattat ttttgatggc gttaactcgg cgtttcatct gtggtgcaac
     1441 gggcgctggg tcggttacgg ccaggacagt cgtttgccgt ctgaatttga cctgagcgca
     1501 tttttacgcg ccggagaaaa ccgcctcgcg gtgatggtgc tgcgctggag tgacggcagt
     1561 tatctggaag atcaggatat gtggcggatg agcggcattt tccgtgacgt ctcgttgctg
     1621 cataaaccga ctacacaaat cagcgatttc catgttgcca ctcgctttaa tgatgatttc
     1681 agccgcgctg tactggaggc tgaagttcag atgtgcggcg agttgcgtga ctacctacgg
     1741 gtaacagttt ctttatggca gggtgaaacg caggtcgcca gcggcaccgc gcctttcggc
     1801 ggtgaaatta tcgatgagcg tggtggttat gccgatcgcg tcacactacg tctgaacgtc
     1861 gaaaacccga aactgtggag cgccgaaatc ccgaatctct atcgtgcggt ggttgaactg
     1921 cacaccgccg acggcacgct gattgaagca gaagcctgcg atgtcggttt ccgcgaggtg
     1981 cggattgaaa atggtctgct gctgctgaac ggcaagccgt tgctgattcg aggcgttaac
     2041 cgtcacgagc atcatcctct gcatggtcag gtcatggatg agcagacgat ggtgcaggat
     2101 atcctgctga tgaagcagaa caactttaac gccgtgcgct gttcgcatta tccgaaccat
     2161 ccgctgtggt acacgctgtg cgaccgctac ggcctgtatg tggtggatga agccaatatt
     2221 gaaacccacg gcatggtgcc aatgaatcgt ctgaccgatg atccgcgctg gctaccggcg
     2281 atgagcgaac gcgtaacgcg aatggtgcag cgcgatcgta atcacccgag tgtgatcatc
     2341 tggtcgctgg ggaatgaatc aggccacggc gctaatcacg acgcgctgta tcgctggatc
     2401 aaatctgtcg atccttcccg cccggtgcag tatgaaggcg gcggagccga caccacggcc
     2461 accgatatta tttgcccgat gtacgcgcgc gtggatgaag accagccctt cccggctgtg
     2521 ccgaaatggt ccatcaaaaa atggctttcg ctacctggag agacgcgccc gctgatcctt
     2581 tgcgaatacg cccacgcgat gggtaacagt cttggcggtt tcgctaaata ctggcaggcg
     2641 tttcgtcagt atccccgttt acagggcggc ttcgtctggg actgggtgga tcagtcgctg
     2701 attaaatatg atgaaaacgg caacccgtgg tcggcttacg gcggtgattt tggcgatacg
     2761 ccgaacgatc gccagttctg tatgaacggt ctggtctttg cgcgatcgca cctcagcgtc
     2821 gtgactggga aaaccctggc gactagtctt ggactcctgt tgatagatcc agtaatgacc
     2881 tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt ttttattggt
     2941 gagaatccag acgtgtaggc tggagctgct tccctgcagg agtcaggtag acgagaaacg
     3001 ttccgtccgt ctgggtcagt tgggcaaagt tgatgaccgg gtcgtccgtt ctgtgcaaca
     3061 ggtgacatca ctagagtcta gggacctctt tagctccttg gaagctgtca gtagtatacc
     3121 taataattta tctacattcc ctttagtaac gtgtaacttt ccaaatttac aaaagcgact
     3181 catagaatta tttcctcccg ttaaataata gataactatt aaaaatagac aatacttgct
     3241 cataagtaac ggtacttaaa ttgtttactt tggcgtgttt cattgcttga tgaaactgat
     3301 ttttagtaaa cagttgacga tattctcgat tgacccattt tgaaacaaag tacgtatata
     3361 gcttccaata tttatctgga acatctgtgg tatggcgggt aagttttatt aagacactgt
     3421 ttacttttgg tttaggatga aagcattccg ctggcagctt aagcaattgc tgaatcgaga
     3481 cttgagtgtg caagagcaac cctagtgttc ggtgaatatc caaggtacgc ttgtagaatc
     3541 cttcttcaac aatcagatag atgtcagacg catggctttc aaaaaccact tttttaataa
     3601 tttgtgtgct taaatggtaa ggaatactcc caacaatttt atacctctgt ttgttaggga
     3661 attgaaactg tagaatatct tggtgaatta aagtgacacg agtattcagt tttaattttt
     3721 ctgacgataa gttgaataga tgactgtcta attcaataga cgttacctgt ttacttattt
     3781 tagccagttt cgtcgttaaa tgccctttac ctgttccaat ttcgtaaacg gtatcggttt
     3841 cttttaaatt caattgtttt attatttggt tgagtacttt ttcactcgtt aaaaagtttt
     3901 gagaatattt tatatttttg ttcatgtaat cactccttct taattacaaa tttttagcat
     3961 ctaatttaac ttcaattcct attatacaaa attttaagat actgcactat caacacactc
     4021 ttaagtttgc ttctaagtct tatttccata acttctttta cgtttccgcc attctttgct
     4081 gtttcgattt ttatgatatg gtgcaagtca gcacgaacac gaaccgtctt atctcccatt
     4141 atatcttttt ttgcactgat tggtgtatca tttcgttttt ctttttgtgc tagaggatca
     4201 attcttgaag acgaaagggc ctcgtgatac gcgttgtcct tttccgctgt gttataaacc
     4261 tgtgtgagag ttaagtttac atgcctaacc ttaactttta cgcaggttca gcttatgcaa
     4321 gctggtaccc taaggaggat attcatatgc aagcacttca gcgtttcttt ggaatatgtc
     4381 ctgactccgc atattgaaac acctgcaagg aaggcagcgg gtgtccatta aaatcaaata
     4441 aagcttgatt atctacagca cttcccccat accacttccc ggcgtcttca gggtcgtatt
     4501 cagcagcata gctggacgcc caccctgacc cgtatgtttc ccataacact ttgtttttct
     4561 ctatctgtgt cttcggcccg actggaatcc acgccggctc ccagtagaaa acaccaagtc
     4621 ctgctttgcc cgtattcgcc actgcctcca ttacatctct tactgcagtt gcctggcctt
     4681 gaacagaaat tggatatggc aacgtctgcc cgctttttgg tgctgtattt ccatgcccat
     4741 cgccatcctc agcggtatag gtgtacgatg tctccgccac catgactttt ttgccgtacg
     4801 tattggcaac agccttcagc acggaggtca aattttgtaa tgtgccatgc cagaaaggat
     4861 aataggagct agcaaacaca tcataatcca ctttgttttt gctgagtgtt tctgcaataa
     4921 atgaatacct tccagccgtt tcaggattgg taaaatgcag ggcgaccaaa atatttgaat
     4981 ttgtctccct gaccgctcgg cttccttcat taaataattg gcacatcttc gtccaatcag
     5041 tctcaccggc aaatcacctg actgggtccc caataattac gatttaaatt agtagcccgc
     5101 ctaatgagcg ggcttttttt taattcccct atttgtttat ttttctaaat acattcaaat
     5161 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5221 agtatgagca ttcagcattt tcgtgtggcg ctgattccgt tttttgcggc gttttgcctg
     5281 ccggtgtttg cgcatccgga aaccctggtg aaagtgaaag atgcggaaga tcaactgggt
     5341 gcgcgcgtgg gctatattga actggatctg aacagcggca aaattctgga atcttttcgt
     5401 ccggaagaac gttttccgat gatgagcacc tttaaagtgc tgctgtgcgg tgcggttctg
     5461 agccgtgtgg atgcgggcca ggaacaactg ggccgtcgta ttcattatag ccagaacgat
     5521 ctggtggaat atagcccggt gaccgaaaaa catctgaccg atggcatgac cgtgcgtgaa
     5581 ctgtgcagcg cggcgattac catgagcgat aacaccgcgg cgaacctgct gctgacgacc
     5641 attggcggtc cgaaagaact gaccgcgttt ctgcataaca tgggcgatca tgtgacccgt
     5701 ctggatcgtt gggaaccgga actgaacgaa gcgattccga acgatgaacg tgataccacc
     5761 atgccggcag caatggcgac caccctgcgt aaactgctga cgggtgagct gctgaccctg
     5821 gcaagccgcc agcaactgat tgattggatg gaagcggata aagtggcggg tccgctgctg
     5881 cgtagcgcgc tgccggctgg ctggtttatt gcggataaaa gcggtgcggg cgaacgtggc
     5941 agccgtggca ttattgcggc gctgggcccg gatggtaaac cgagccgtat tgtggtgatt
     6001 tataccaccg gcagccaggc gacgatggat gaacgtaacc gtcagattgc ggaaattggc
     6061 gcgagcctga ttaaacattg gtaaaccgat acaattaaag gctccttttg gagccttttt
     6121 ttttggacga cccttgtcct tttccgctgc ataaccctgc ttcggggtca ttatagcgat
     6181 tttttcggta tatccatcct ttttcgcacg atatacagga ttttgccaaa gggttcgtgt
     6241 agactttcct tggtgtatcc aacggcgtca gccgggcagg ataggtgaag taggcccacc
     6301 cgcgagcggg tgttccttct tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa
     6361 tcctgctctg cgaggctggc cgtaggccgg ccctaccggc gcggcagcgt tacccgtgtc
     6421 ggcggctcca acggctcgcc atcgtccaga aaacacggct catcgggcat cggcaggcgc
     6481 tgctgcccgc gccgttccca ttcctccgtt tcggtcaagg ctggcaggtc tggttccatg
     6541 cccggaatgc cgggctggct gggcggctcc tcgccggggc cggtcggtag ttgctgctcg
     6601 cccggataca gggtcgggat gcggcgcagg tcgccatgcc ccaacagcga ttcgtcctgg
     6661 tcgtcgtgat caaccaccac ggcggcactg aacaccgaca ggcgcaactg gtcgcggggc
     6721 tggccccacg ccacgcggtc attgaccacg taggccgaca cggtgccggg gccgttgagc
     6781 ttcacgacgg agatccagcg ctcggccacc aagtccttga ctgcgtattg gaccgtccgc
     6841 aaagaacgtc cgatgagctt ggaaagtgtc ttctggctga ccaccacggc gttctggtgg
     6901 cccatctgcg ccacgaggtg atgcagcagc attgccgccg tgggtttcct cgcaataagc
     6961 ccggcccacg cctcatgcgc tttgcgttcc gtttgcaccc agtgaccggg cttgttcttg
     7021 gcttgaatgc cgatttctct ggactgcgtg gccatgctta tctccatgcg gtaggggtgc
     7081 cgcacggttg cggcaccatg cgcaatcagc tgcaactttt cggcagcgcg acaacaatta
     7141 tgcgttgcgt aaaagtggca gtcaattaca gattttcttt aacctacgca atgagctatt
     7201 gcggggggtg ccgcaatgag ctgttgcgta cccccctttt ttaagttgtt gatttttaag
     7261 tctttcgcat ttcgccctat atctagttct ttggtgccca aagaagggca cccctgcggg
     7321 gttcccccac gccttcggcg cggctccccc tccggcaaaa agtggcccct ccggggcttg
     7381 ttgatcgact gcgcggcctt cggccttgcc caaggtggcg ctgccccctt ggaacccccg
     7441 cactcgccgc cgtgaggctc ggggggcagg cgggcgggct tcgcccttcg actgccccca
     7501 ctcgcatagg cttgggtcgt tccaggcgcg tcaaggccaa gccgctgcgc ggtcgctgcg
     7561 cgagccttga cccgccttcc acttggtgtc caaccggcaa gcgaagcgcg caggccgcag
     7621 gccggaggct tttccccaga gaaaattaaa aaaattgatg gggcaaggcc gcaggccgcg
     7681 cagttggagc cggtgggtat gtggtcgaag gctgggtagc cggtgggcaa tccctgtggt
     7741 caagctcgtg ggcaggcgca gcctgtccat cagcttgtcc agcagggttg tccacgggcc
     7801 gagcgaagcg agccagccgg tggccgctcg cggccatcgt ccacatatcc acgggctggc
     7861 aagggagcgc agcgaccgcg cagggcgaag cccggagagc aagcccgtag ggggaggcga
     7921 tctcgagagc ttgcatactc gccagaaaga tccagaaaaa ctcgttgact gaaccgattg
     7981 taaacgatca ggttcagatt gactcttatt acaacgcatc gagtgaattg tcaggtgatc
     8041 tttacggata ttatcaaatc gatgaacacc gttacggcat tattatcctt gatgtgatgg
     8101 ggcacggaat ttcatctgca ttgattacga tgtctcttca tcctttgttc cagcgccaga
     8161 tcacgcaagg tctcagcccc gttaaagtta tgaaggaatt ggaccggcac cttcacagct
     8221 tgtttcaaaa tgatgaggaa gcaagacatt attgcactgc tatttatctc gagattgata
     8281 tagcacggca aagaattgat tatgtcaacg cagggcatcc gccggcactt tggcaggacg
     8341 atagcggaac tcaacatctg cttcacgcga cctcgcctcc gatcgggatg tttgaagact
     8401 tagagtttca gtcaagcagc ctttcataca cggaggacgg aagattgctt ctgtatacgg
     8461 atggcgtgat ggatcctacg gcttcatgtt atttatttga tttactaaag gatcatcctg
     8521 attcgccaat cgctgacctg aaggagaaaa tccttacatc actgcagcat caaaaggaag
     8581 ctcatcataa gagtgatgac gaatgtttta tattagttga tgtaaaataa aatgatccat
     8641 gagacataac aatgatttgt tacatctcat
//
