LOCUS       sequence-365732-        8859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli - B. subtilis nicking vector pProUSER13E3F. Ap-BBR1; 
            yhgE-insertion-site; Spc; tetR-Ptet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8859)
  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 8859)
  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: pProUSER13E3F
FEATURES             Location/Qualifiers
     source          1..8859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(46..132)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     147..169
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             complement(183..809)
                     /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     714..733
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    916..934
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     terminator      2844..2938
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        5244..5335
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5336..5404,5405..6196)
                     /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      6200..6239
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            6386..6494
                     /note="incP origin of transfer"
     CDS             complement(6505..7167)
                     /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      7168..7939
                     /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 cataaaccag cttgtacagg cgcgcccagc tgtctagggc ggcggatttg tcctactcag
       61 gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt
      121 tcgttttatt tgatgccttt aattaaagcg gataacaatt tcacacagga ggccgatcgc
      181 gtttaagacc cactttcaca tttaagttgt ttttctaatc cgcatatgat caattcaagg
      241 ccgaataaga aggctggctc tgcaccttgg tgatcaaata attcgatagc ttgtcgtaat
      301 aatggcggca tactatcagt agtaggtgtt tccctttctt ctttagcgac ttgatgctct
      361 tgatcttcca atacgcaacc taaagtaaaa tgccccacag cgctgagtgc atataatgca
      421 ttctctagtg aaaaaccttg ttggcataaa aaggctaatt gattttcgag agtttcatac
      481 tgtttttctg taggccgtgt acctaaatgt acttttgctc catcgcgatg acttagtaaa
      541 gcacatctaa aacttttagc gttattacgt aaaaaatctt gccagctttc cccttctaaa
      601 gggcaaaagt gagtatggtg cctatctaac atctcaatgg ctaaggcgtc gagcaaagcc
      661 cgcttatttt ttacatgcca atacaatgta ggctgctcta cacctagctt ctgggcgagt
      721 ttacgggttg ttaaaccttc gattccgacc tcattaagca gctctaatgc gctgttaatc
      781 actttacttt tatctaatct agacatcatt aattcctcct ttttgttgac attatatcat
      841 tgatagagtt atttgtcaaa ctagattttt atttcgatgc cctggacttc atgaaaaact
      901 aaaaaaaata ttgacactct atcattgata gagtataatt aaaataagct tgatcgtagc
      961 gttaacagat ctgagctcct gcagtaagct gaggcgatcg caacgtcgtg actgggaaaa
     1021 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     1081 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     1141 gcgctttgcc tggtttccgg caccagaagc ggtgccggaa agctggctgg agtgcgatct
     1201 tcctgaggcc gatactgtcg tcgtcccctc aaactggcag atgcacggtt acgatgcgcc
     1261 catctacacc aacgtgacct atcccattac ggtcaatccg ccgtttgttc ccacggagaa
     1321 tccgacgggt tgttactcgc tcacatttaa tgttgatgaa agctggctac aggaaggcca
     1381 gacgcgaatt atttttgatg gcgttaactc ggcgtttcat ctgtggtgca acgggcgctg
     1441 ggtcggttac ggccaggaca gtcgtttgcc gtctgaattt gacctgagcg catttttacg
     1501 cgccggagaa aaccgcctcg cggtgatggt gctgcgctgg agtgacggca gttatctgga
     1561 agatcaggat atgtggcgga tgagcggcat tttccgtgac gtctcgttgc tgcataaacc
     1621 gactacacaa atcagcgatt tccatgttgc cactcgcttt aatgatgatt tcagccgcgc
     1681 tgtactggag gctgaagttc agatgtgcgg cgagttgcgt gactacctac gggtaacagt
     1741 ttctttatgg cagggtgaaa cgcaggtcgc cagcggcacc gcgcctttcg gcggtgaaat
     1801 tatcgatgag cgtggtggtt atgccgatcg cgtcacacta cgtctgaacg tcgaaaaccc
     1861 gaaactgtgg agcgccgaaa tcccgaatct ctatcgtgcg gtggttgaac tgcacaccgc
     1921 cgacggcacg ctgattgaag cagaagcctg cgatgtcggt ttccgcgagg tgcggattga
     1981 aaatggtctg ctgctgctga acggcaagcc gttgctgatt cgaggcgtta accgtcacga
     2041 gcatcatcct ctgcatggtc aggtcatgga tgagcagacg atggtgcagg atatcctgct
     2101 gatgaagcag aacaacttta acgccgtgcg ctgttcgcat tatccgaacc atccgctgtg
     2161 gtacacgctg tgcgaccgct acggcctgta tgtggtggat gaagccaata ttgaaaccca
     2221 cggcatggtg ccaatgaatc gtctgaccga tgatccgcgc tggctaccgg cgatgagcga
     2281 acgcgtaacg cgaatggtgc agcgcgatcg taatcacccg agtgtgatca tctggtcgct
     2341 ggggaatgaa tcaggccacg gcgctaatca cgacgcgctg tatcgctgga tcaaatctgt
     2401 cgatccttcc cgcccggtgc agtatgaagg cggcggagcc gacaccacgg ccaccgatat
     2461 tatttgcccg atgtacgcgc gcgtggatga agaccagccc ttcccggctg tgccgaaatg
     2521 gtccatcaaa aaatggcttt cgctacctgg agagacgcgc ccgctgatcc tttgcgaata
     2581 cgcccacgcg atgggtaaca gtcttggcgg tttcgctaaa tactggcagg cgtttcgtca
     2641 gtatccccgt ttacagggcg gcttcgtctg ggactgggtg gatcagtcgc tgattaaata
     2701 tgatgaaaac ggcaacccgt ggtcggctta cggcggtgat tttggcgata cgccgaacga
     2761 tcgccagttc tgtatgaacg gtctggtctt tgcgcgatcg cacctcagcg tcgtgactgg
     2821 gaaaaccctg gcgactagtc ttggactcct gttgatagat ccagtaatga cctcagaact
     2881 ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc
     2941 agacgtgtag gctggagctg cttccctgca ggagtcaggt caatcatcag ataactatgg
     3001 cggcacgtgc attaaccacg gttgtatccc gtctaaagta ctcgtctgtg caacaggtga
     3061 catcacgaaa cgatcctcat cctgtctctt gatccatgga ttacgcgtta acccgggccc
     3121 gcggatgcat atgatcagat cttaaggcct aggtctagag gatcgatctg tataataaag
     3181 aataattatt aatctgtaga caaattgtga aaggatgtac ttaaacgcta acggtcagct
     3241 ttattgaaca gtaatttaag tatatgtcca atctagggta agtaaattga gtatcaatat
     3301 aaactttata tgaacataat caacgaggtg aaatcatgag caatttgatt aacggaaaaa
     3361 taccaaatca agcgattcaa acattaaaaa tcgtaaaaga tttatttgga agttcaatag
     3421 ttggagtata tctatttggt tcagcagtaa atggtggttt acgcattaac agcgatgtag
     3481 atgttctagt cgtcgtgaat catagtttac ctcaattaac tcgaaaaaaa ctaacagaaa
     3541 gactaatgac tatatcagga aagattggaa atacggattc tgttagacca cttgaagtta
     3601 cggttataaa taggagtgaa gttgtccctt ggcaatatcc tccaaaaaga gaatttatat
     3661 acggtgagtg gctcaggggt gaatttgaga atggacaaat tcaggaacca agctatgatc
     3721 ctgatttggc tattgtttta gcacaagcaa gaaagaatag tatttctcta tttggtcctg
     3781 attcttcaag tatacttgtc tccgtacctt tgacagatat tcgaagagca attaaggatt
     3841 ctttgccaga actaattgag gggataaaag gtgatgagcg taatgtaatt ttaaccctag
     3901 ctcgaatgtg gcaaacagtg actactggtg aaattacctc gaaagatgtc gctgcagaat
     3961 gggctatacc tcttttacct aaagagcatg taactttact ggatatagct agaaaaggct
     4021 atcggggaga gtgtgatgat aagtgggaag gactatattc aaaggtgaaa gcactcgtta
     4081 agtatatgaa aaattctata gaaacttctc tcaattaggc taattttatt gcaataacag
     4141 gtgcttactt ttctggagtt ctttagcaaa tttttttatt agctgaactt agtattagtg
     4201 gccatactcc tccaatccaa agctatttag aaagattact atatcctcaa acaggcggta
     4261 acgcgttgtc cttttccgct ttaataaact atggaagtat gtacagtctt gcaatgttga
     4321 gtgaacaaac ttccataata aaatatgcaa gctggtaccc taaggaggat attcatatgc
     4381 aagcactaga aaaagaactt gtttccttgg aaataagttc tttttttgtt acatccccca
     4441 tttaataagc aatcctgtat aggttaatcc gccgccaaac ccgaaaagca aaacgatttg
     4501 atcttttttc agcttcccgg ctttcactgc gagatcgagc gccaaaacaa ttgaaaccga
     4561 agacgtgttt ccgtagtgct caacactagt gagcgttttt tcaatcggga acggtgtttt
     4621 ttcacaaatt gactcgatca tgcgcaagtt ggcgctgtga ggaacaaacc aatcgagatc
     4681 atcggagctg agtcctgctt tatgtaaaag ccgttcaaat tcgccaggga cggttcttgc
     4741 ggcccattta tatacctcgc gtccgttttg caccattttt ccggaaccga caagctgcac
     4801 accgtttatt tcatttcgca gtccggcacg atacaaaata tcgccgccgt tcccgcttgt
     4861 tccttgtaca gacgcaagaa atcccggcgt ctcttcatct cgttctacta acagcgcacc
     4921 cgcggcatcg ccgaacagta cgcatgtcgt tcgatcggta taatcggtta cctttgataa
     4981 cgtctctccg gcgatgacga gaattttttg atgaaggcca gatgtgatca atccatttgc
     5041 caaatggagg ccgtatgtca gcccggcgca tgtcgcatta atatccagcg cgccggtgct
     5101 ttcccagccg aaatattcct gtacgcggca tgccgtactc ggaaaggcgt aatcggatgt
     5161 ggttgtgacc tgactgggtc cccaataatt acgatttaaa ttagtagccc gcctaatgag
     5221 cgggcttttt tttaattccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5281 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5341 cattcagcat tttcgtgtgg cgctgattcc gttttttgcg gcgttttgcc tgccggtgtt
     5401 tgcgcatccg gaaaccctgg tgaaagtgaa agatgcggaa gatcaactgg gtgcgcgcgt
     5461 gggctatatt gaactggatc tgaacagcgg caaaattctg gaatcttttc gtccggaaga
     5521 acgttttccg atgatgagca cctttaaagt gctgctgtgc ggtgcggttc tgagccgtgt
     5581 ggatgcgggc caggaacaac tgggccgtcg tattcattat agccagaacg atctggtgga
     5641 atatagcccg gtgaccgaaa aacatctgac cgatggcatg accgtgcgtg aactgtgcag
     5701 cgcggcgatt accatgagcg ataacaccgc agcgaacctg ctgctgacga ccattggcgg
     5761 tccgaaagaa ctgaccgcgt ttctgcataa catgggcgat catgtgaccc gtctggatcg
     5821 ttgggaaccg gaactgaacg aagcgattcc gaacgatgaa cgtgatacca ccatgccggc
     5881 agcaatggcg accaccctgc gtaaactgct gacgggtgag ctgctgaccc tggcaagccg
     5941 ccagcaactg attgattgga tggaagcgga taaagtggcg ggtccgctgc tgcgtagcgc
     6001 gctgccggct ggctggttta ttgcggataa aagcggtgcg ggcgaacgtg gcagccgtgg
     6061 cattattgcg gcgctgggcc cggatggtaa accgagccgt attgtggtga tttataccac
     6121 cggcagccag gcgacgatgg atgaacgtaa ccgtcagatt gcggaaattg gcgcgagcct
     6181 gattaaacat tggtaaaccg atacaattaa aggctccttt tggagccttt ttttttggac
     6241 gacccttgtc cttttccgct gcataaccct gcttcggggt cattatagcg attttttcgg
     6301 tatatccatc ctttttcgca cgatatacag gattttgcca aagggttcgt gtagactttc
     6361 cttggtgtat ccaacggcgt cagccgggca ggataggtga agtaggccca cccgcgagcg
     6421 ggtgttcctt cttcactgtc ccttattcgc acctggcggt gctcaacggg aatcctgctc
     6481 tgcgaggctg gccgtaggcc ggccctaccg gcgcggcagc gttacccgtg tcggcggctc
     6541 caacggctcg ccatcgtcca gaaaacacgg ctcatcgggc atcggcaggc gctgctgccc
     6601 gcgccgttcc cattcctccg tttcggtcaa ggctggcagg tctggttcca tgcccggaat
     6661 gccgggctgg ctgggcggct cctcgccggg gccggtcggt agttgctgct cgcccggata
     6721 cagggtcggg atgcggcgca ggtcgccatg ccccaacagc gattcgtcct ggtcgtcgtg
     6781 atcaaccacc acggcggcac tgaacaccga caggcgcaac tggtcgcggg gctggcccca
     6841 cgccacgcgg tcattgacca cgtaggccga cacggtgccg gggccgttga gcttcacgac
     6901 ggagatccag cgctcggcca ccaagtcctt gactgcgtat tggaccgtcc gcaaagaacg
     6961 tccgatgagc ttggaaagtg tcttctggct gaccaccacg gcgttctggt ggcccatctg
     7021 cgccacgagg tgatgcagca gcattgccgc cgtgggtttc ctcgcaataa gcccggccca
     7081 cgcctcatgc gctttgcgtt ccgtttgcac ccagtgaccg ggcttgttct tggcttgaat
     7141 gccgatttct ctggactgcg tggccatgct tatctccatg cggtaggggt gccgcacggt
     7201 tgcggcacca tgcgcaatca gctgcaactt ttcggcagcg cgacaacaat tatgcgttgc
     7261 gtaaaagtgg cagtcaatta cagattttct ttaacctacg caatgagcta ttgcgggggg
     7321 tgccgcaatg agctgttgcg tacccccctt ttttaagttg ttgattttta agtctttcgc
     7381 atttcgccct atatctagtt ctttggtgcc caaagaaggg cacccctgcg gggttccccc
     7441 acgccttcgg cgcggctccc cctccggcaa aaagtggccc ctccggggct tgttgatcga
     7501 ctgcgcggcc ttcggccttg cccaaggtgg cgctgccccc ttggaacccc cgcactcgcc
     7561 gccgtgaggc tcggggggca ggcgggcggg cttcgccctt cgactgcccc cactcgcata
     7621 ggcttgggtc gttccaggcg cgtcaaggcc aagccgctgc gcggtcgctg cgcgagcctt
     7681 gacccgcctt ccacttggtg tccaaccggc aagcgaagcg cgcaggccgc aggccggagg
     7741 cttttcccca gagaaaatta aaaaaattga tggggcaagg ccgcaggccg cgcagttgga
     7801 gccggtgggt atgtggtcga aggctgggta gccggtgggc aatccctgtg gtcaagctcg
     7861 tgggcaggcg cagcctgtcc atcagcttgt ccagcagggt tgtccacggg ccgagcgaag
     7921 cgagccagcc ggtggccgct cgcggccatc gtccacatat ccacgggctg gcaagggagc
     7981 gcagcgaccg cgcagggcga agcccggaga gcaagcccgt agggggaggc gatctcgaga
     8041 gcttgcatga cgggtctaaa caggtccaaa gcggatcatc aaaacttgca gacggatctg
     8101 catcacttga tacaggttta ggcaagctat tggacggcac aggagagctg tctagtaagc
     8161 tgaaggatgc ggctgaccaa acaggagata tcgatgctga tgatcaaaca tacggcatgt
     8221 ttgcggatcc ggttaaaacc aaagatgatg ccattcattc cgttcctaac tatggaacag
     8281 ggctgacacc ttatatcctg tcaatgggct tgtatgtcgg cggcatcatg ctgacggttg
     8341 tcttcccgct gaaggaagcg tcaggacgtc cgagaaacgg ttttgagtgg ttcttcagca
     8401 aattcaatgt catgatgctt gtaggaatca ttcaatcatt gattgtggcg acggttcttc
     8461 tattgggaat tggtcttgag gtagagagca cgtggagatt ttacgtgttt acgatcatta
     8521 cgagtctcgc tttcctggcg ataattcaat ttttggctac aacgatgggc aacccggggc
     8581 gatttatcgc agtcattatt ttagtgctgc agctgggagc gagcggagga accttcccgc
     8641 tggaattgct tccgaatttc tatcaggtca tccacggcgc attgccgatg acctacagca
     8701 ttaacggatt cagagcagtc atttcaaacg gtgactttgg ctacatgtgg cagatggccg
     8761 gcgtcttaat cggcattgcc ctcgtcatga ttgcactaag cattacttac tttacaatgt
     8821 taagccggaa ggaagaaaca tcagaagaac agccggctt
//
