LOCUS       sequence-466831-        6346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BEVA Golden Gate cloning vector; BEVA2.0 Sucrose curable broad host 
            range plasmid . L1-pBBR1-Sp-sacB-ELT4; GG assembly from pOGG004, 
            pNDGG002, pOGG011, pNDGG008, and pOGG014, SpR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6346)
  AUTHORS   Geddes BA, Williamson R, Schumacher J, Ardi A, Levin G, Cervenka E, 
            Huang R, diCenzo G
  TITLE     BEVA2.0: modular assembly of golden gate-compatible vectors with 
            expanded utility for genetic engineering.
  JOURNAL   Can J Microbiol. 2025 Jan 1;71:1-13. doi: 10.1139/cjm-2024-0246.
  PUBMED    39919271
REFERENCE   2  (bases 1 to 6346)
  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: "Can J 
            Microbiol."; date: "2025-01-1"; volume: "71:1-13. doi"; pages: " 
            10.1139/cjm-2024-0246"
COMMENT     Sequence Label: pNDGG070
FEATURES             Location/Qualifiers
     source          1..6346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            140..248
                     /note="incP origin of transfer"
     CDS             complement(259..921)
                     /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      922..1693
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     promoter        1785..2230
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(2231..2317,2318..3652)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVSEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     terminator      complement(3746..3832)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    3920..3941
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3956..3961,3962..3979,3980..3986)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3994..4010
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3999..4021
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4018..4034
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4018..4034
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4030..4353
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    4047..4103
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(4104..4121)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4104..4120)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4113..4135)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4329..4348)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     terminator      4458..4552
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5296..6087
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 actacttttc cgctgcataa ccctgcttcg gggtcattat agcgattttt tcggtatatc
       61 catccttttt cgcacgatat acaggatttt gccaaagggt tcgtgtagac tttccttggt
      121 gtatccaacg gcgtcagccg ggcaggatag gtgaagtagg cccacccgcg agcgggtgtt
      181 ccttcttcac tgtcccttat tcgcacctgg cggtgctcaa cgggaatcct gctctgcgag
      241 gctggccgta ggccggccct accggcgcgg cagcgttacc cgtgtcggcg gctccaacgg
      301 ctcgccatcg tccagaaaac acggctcatc gggcatcggc aggcgctgct gcccgcgccg
      361 ttcccattcc tccgtttcgg tcaaggctgg caggtctggt tccatgcccg gaatgccggg
      421 ctggctgggc ggctcctcgc cggggccggt cggtagttgc tgctcgcccg gatacagggt
      481 cgggatgcgg cgcaggtcgc catgccccaa cagcgattcg tcctggtcgt cgtgatcaac
      541 caccacggcg gcactgaaca ccgacaggcg caactggtcg cggggctggc cccacgccac
      601 gcggtcattg accacgtagg ccgacacggt gccggggccg ttgagcttca cgacggagat
      661 ccagcgctcg gccaccaagt ccttgactgc gtattggacc gtccgcaaag aacgtccgat
      721 gagcttggaa agagtctttt ggctgaccac cacggcgttc tggtggccca tctgcgcgac
      781 gaggtgatgc agcagcattg ccgccgtggg tttcctcgca ataagcccgg cccacgcctc
      841 atgcgctttg cgttccgttt ggacccagtg accgggcttg ttcttggctt gaatgccgat
      901 ttctctggac tgcgtggcca tgcttatctc catgcggtag gggtgccgca cggttgcggc
      961 accatgcgca atcagctgca acttttcggc agcgcgacaa caattatgcg ttgcgtaaaa
     1021 gtggcagtca attacagatt ttctttaacc tacgcaatga gctattgcgg ggggtgccgc
     1081 aatgagctgt tgcgtacccc ccttttttaa gttgttgatt tttaagtctt tcgcatttcg
     1141 ccctatatct agttctttgg tgcccaaaga agggcacccc tgcggggttc ccccacgcct
     1201 tcggcgcggc tccccctccg gcaaaaagtg gcccctccgg ggcttgttga tcgactgcgc
     1261 ggccttcggc cttgcccaag gtggcgctgc ccccttggaa cccccgcact cgccgccgtg
     1321 aggctcgggg ggcaggcggg cgggcttcgc ccttcgactg cccccactcg cataggcttg
     1381 ggtcgttcca ggcgcgtcaa ggccaagccg ctgcgcggtc gctgcgcgag ccttgacccg
     1441 ccttcctctt ggtgtccaac cggcaagcga agcgcgcagg ccgcaggccg gaggcttttc
     1501 cccagagaaa attaaaaaaa ttgatggggc aaggccgcag gccgcgcagt tggagccggt
     1561 gggtatgtgg tcgaaggctg ggtagccggt gggcaatccc tgtggtcaag ctcgtgggca
     1621 ggcgcagcct gtccatcagc ttgtccagca gggttgtcca cgggccgagc gaagcgagcc
     1681 agccggtggc cgctcgcggc catcgtccac atatccacgg gctggcaagg gagcgcagcg
     1741 accgcgcagg gcgaagcccg gagagcaagc ccgtaggggg ttaccacata tacctgccgt
     1801 tcactattat ttagtgaaat gagatattat gatattttct gaattgtgat taaaaaggca
     1861 actttatgcc catgcaacag aaactataaa aaatacagag aatgaaaaga aacagataga
     1921 ttttttagtt ctttaggccc gtagtctgca aatcctttta tgattttcta tcaaacaaaa
     1981 gaggaaaata gaccagttgc aatccaaacg agagtctaat agaatgaggt cgaaaagtaa
     2041 atcgcgcggg tttgttactg ataaagcagg caagacctaa aatgtgtaaa gggcaaagtg
     2101 tatactttgg cgtcacccct tacatatttt aggtcttttt ttattgtgcg taactaactt
     2161 gccatcttca aacaggaggg ctggaagaag cagaccgcta acacagtaca taaaaaagga
     2221 gacatgaacg atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac
     2281 cgcactgctg gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc aaaagccata
     2341 taaggaaaca tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca
     2401 gcaaaaaaat gaaaaatatc aagtttctga atttgattcg tccacaatta aaaatatctc
     2461 ttctgcaaaa ggcctggacg tttgggacag ctggccatta caaaacgctg acggcactgt
     2521 cgcaaactat cacggctacc acatcgtctt tgcattagcc ggagatccta aaaatgcgga
     2581 tgacacatcg atttacatgt tctatcaaaa agtcggcgaa acttctattg acagctggaa
     2641 aaacgctggc cgcgtcttta aagacagcga caaattcgat gcaaatgatt ctatcctaaa
     2701 agaccaaaca caagaatggt caggttcagc cacatttaca tctgacggaa aaatccgttt
     2761 attctacact gatttctccg gtaaacatta cggcaaacaa acactgacaa ctgcacaagt
     2821 taacgtatca gcatcagaca gctctttgaa catcaacggt gtagaggatt ataaatcaat
     2881 ctttgacggt gacggaaaaa cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta
     2941 cagctcaggc gacaaccata cgctgagaga tcctcactac gtagaagata aaggccacaa
     3001 atacttagta tttgaagcaa acactggaac tgaagatggc taccaaggcg aagaatcttt
     3061 atttaacaaa gcatactatg gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact
     3121 tctgcaaagc gataaaaaac gcacggctga gttagcaaac ggcgctctcg gtatgattga
     3181 gctaaacgat gattacacac tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt
     3241 aacagatgaa attgaacgcg cgaacgtctt taaaatgaac ggcaaatggt atctgttcac
     3301 tgactcccgc ggatcaaaaa tgacgattga cggcattacg tctaacgata tttacatgct
     3361 tggttatgtt tctaattctt taactggccc atacaagccg ctgaacaaaa ctggccttgt
     3421 gttaaaaatg gatcttgatc ctaacgatgt aacctttact tactcacact tcgctgtacc
     3481 tcaagcgaaa ggaaacaatg tcgtgattac aagctatatg acaaacagag gattctacgc
     3541 agacaaacaa tcaacgtttg cgccgagctt cctgctgaac atcaaaggca agaaaacatc
     3601 tgttgtcaaa gacagcatcc ttgaacaagg acaattaaca gttaataaat aaaaacgcaa
     3661 aagaaatgcc gatgggtacg tacccgctct tggactcctg ttactccgga tccgaattcg
     3721 gcaggcatca gatctagggc ggcggatttg tcctactcag gagagcgttc accgacaaac
     3781 aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctgc
     3841 cgaattcgga tccggagtga gaccgcagct ggcacgacag gtttgccgac tggaaagcgg
     3901 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     3961 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     4021 gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcaggtcgac tctagaggat
     4081 ccccgggtac cgagctcgaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     4141 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     4201 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     4261 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc
     4321 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     4381 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4441 acggtctcac gctcttggac tcctgttgat agatccagta atgacctcag aactccatct
     4501 ggatttgttc agaacgctcg gttgccgccg ggcgtttttt attggtgaga atccaggcaa
     4561 ccggatcaat tcccctgctc gcgcaggctg ggtgccaagc tctcgggtaa catcaaggcc
     4621 cgatccttgg agcccttgcc ctcccgcacg atgatcgtgc cgtgatcgaa atccagatcc
     4681 ttgacccgca gttgcaaacc ctcactgatc cgcatgcccg ttccatacag aagctgggcg
     4741 aacaaacgat gctcgccttc cagaaaaccg aggatgcgaa ccacttcatc cggggtcagc
     4801 accaccggca agcgccgcga cggccgaggt cttgcgatct cctgaagcca gggcagatcc
     4861 gtgcacagca ccttgccgta gaagaacagc aaggccgcca atgcctgacg atgcgtggag
     4921 agcgaaacct tgcgctcgtt cgccagccag gacagaaatg cctcgacttc gctgctgccc
     4981 aaggttgccg ggtgacgcac accgtggaaa cggatgaagg cacgaaccca gtggacataa
     5041 gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga
     5101 accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac
     5161 tgtttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg
     5221 ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa
     5281 acaaagttaa acatcatgag ggaagcggtg atcgccgaag tatcgactca actatcagag
     5341 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc
     5401 tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc
     5461 gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct
     5521 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac
     5581 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat
     5641 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg
     5701 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat
     5761 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg
     5821 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac
     5881 agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc
     5941 ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa
     6001 gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc
     6061 gagatcacca aggtagtcgg caaataatgt ctaacaattc gttcaagccg acgccgcttc
     6121 gcggcgcggc ttaactcaag cgttagatgc actaagcaca taattgctca cagccaaact
     6181 atcaggtcaa gtctgctttt attattttta agcgtgcata ataagcccta cacaaattgg
     6241 gagatatatc atgaaaggct ggctttttct tgttatcgca atagttggcg aagtaatcgc
     6301 aacatccgca ttaaaatcta gcgagggctt tactaagctg atccgg
//
