LOCUS       sequence-249595-        4625 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EMMA Kit # 1000000119 extension.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4625)
  TITLE     Lewin Lab Plasmids
REFERENCE   2  (bases 1 to 4625)
  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"
COMMENT     Sequence Label: pRDAX-EMMI-009   BsaI//(A)-AarI-SacB-AarI-(Z)/...
FEATURES             Location/Qualifiers
     source          1..4625
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(6..593)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(86..105)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(615..644)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     promoter        750..1195
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(1196..1282,1283..2617)
                     /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
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     CDS             2715..3374
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2781..2800)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3618..3649
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        3650..3752
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3753..4568
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3826..3845)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      4592..4619
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
ORIGIN
        1 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgatg ctcaagtcag
       61 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      121 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      181 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      241 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
      301 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
      361 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
      421 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
      481 gttacctcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
      541 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
      601 ctttgatttt ctaccgaaga aaggcccacc cgtgaaggtg agccagtgag ttgattgcag
      661 tccagttacg ctggagtctg aggctcgtcc tgaatgggtc tctatatgca ggtgtcgcag
      721 cggacacttc gcgatccttt ttaacccatc acatatacct gccgttcact attatttagt
      781 gaaatgagat attatgatat tttctgaatt gtgattaaaa aggcaacttt atgcccatgc
      841 aacagaaact ataaaaaata cagagaatga aaagaaacag atagattttt tagttcttta
      901 ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa caaaagagga aaatagacca
      961 gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa agtaaatcgc gcgggtttgt
     1021 tactgataaa gcaggcaaga cctaaaatgt gtaaagggca aagtgtatac tttggcgtca
     1081 ccccttacat attttaggtc tttttttatt gtgcgtaact aacttgccat cttcaaacag
     1141 gagggctgga agaagcagac cgctaacaca gtacataaaa aaggagacat gaacgatgaa
     1201 catcaaaaag tttgcaaaac aagcaacagt attaaccttt actaccgcac tgctggcagg
     1261 aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag ccatataagg aaacatacgg
     1321 catttcccat attacacgcc atgatatgct gcaaatccct gaacagcaaa aaaatgaaaa
     1381 atatcaagtt cctgaattcg attcgtccac aattaaaaat atctcttctg caaaaggcct
     1441 ggacgtttgg gacagctggc cattacaaaa cgctgacggc actgtcgcaa actatcacgg
     1501 ctaccacatc gtctttgcat tagccggaga tcctaaaaat gcggatgaca catcgattta
     1561 catgttctat caaaaagtcg gcgaaacttc tattgacagc tggaaaaacg ctggccgcgt
     1621 ctttaaagac agcgacaaat tcgatgcaaa tgattctatc ctaaaagacc aaacacaaga
     1681 atggtcaggt tcagccacat ttacatctga cggaaaaatc cgtttattct acactgattt
     1741 ctccggtaaa cattacggca aacaaacact gacaactgca caagttaacg tatcagcatc
     1801 agacagctct ttgaacatca acggtgtaga ggattataaa tcaatctttg acggtgacgg
     1861 aaaaacgtat caaaatgtac agcagttcat cgatgaaggc aactacagct caggcgacaa
     1921 ccatacgctg agagatcctc actacgtaga agataaaggc cacaaatact tagtatttga
     1981 agcaaacact ggaactgaag atggctacca aggcgaagaa tctttattta acaaagcata
     2041 ctatggcaaa agcacatcat tcttccgtca agaaagtcaa aaacttctgc aaagcgataa
     2101 aaaacgcacg gctgagttag caaacggcgc tctcggtatg attgagctaa acgatgatta
     2161 cacactgaaa aaagtgatga aaccgctgat tgcatctaac acagtaacag atgaaattga
     2221 acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg ttcactgact cccgcggatc
     2281 aaaaatgacg attgacggca ttacgtctaa cgatatttac atgcttggtt atgtttctaa
     2341 ttctttaact ggcccataca agccgctgaa caaaactggc cttgtgttaa aaatggatct
     2401 tgatcctaac gatgtaacct ttacttactc acacttcgct gtacctcaag cgaaaggaaa
     2461 caatgtcgtg attacaagct atatgacaaa cagaggattc tacgcagaca aacaatcaac
     2521 gtttgcgcca agcttcctgc tgaacatcaa aggcaagaaa acatctgttg tcaaagacag
     2581 catccttgaa caaggacaat taacagttaa caaataaaaa cgcaaaagaa aatgccgata
     2641 tcctattggc attttctttt atttcttatc aacataaagg tgaatcccat agggcaggag
     2701 ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat
     2761 ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga
     2821 ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt
     2881 atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg
     2941 caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc
     3001 atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt
     3061 ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta
     3121 aagggtttat tgagaatatg tttttcgtgt cagccaatcc ctgggtgagt ttcaccagtt
     3181 ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat
     3241 attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt
     3301 gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc
     3361 agggcggggc gtaattcccg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct
     3421 gctggagatg gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt
     3481 tctccgcaag aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg
     3541 ccggcttcca tgctcggaaa gccgcacgac acctgcatat agagaccttt aaatcatacc
     3601 tgacctccat agcagaaagt caaaagcctc cgaccggagg cttttgactt gatcggcacg
     3661 taagaggttc caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt
     3721 tatcgagatt ttcaggagct aaggaagcta aaatgagcca tattcaacgg gaaacgtctt
     3781 gctcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc
     3841 gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc
     3901 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg
     3961 tcaggctaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta
     4021 ctcctgatga tgcatggtta ctcaccactg cgatcccagg gaaaacagca ttccaggtat
     4081 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc
     4141 ggttgcattc gattcctgtt tgtaattgtc cttttaacgg cgatcgcgta tttcgcctcg
     4201 ctcaggcgca atcacgaatg aataacggtt tggttggtgc gagtgatttt gatgacgagc
     4261 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac
     4321 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga
     4381 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg
     4441 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa
     4501 aatatggtat tgataatcct gatatgaata aattgcagtt tcacttgatg ctcgatgagt
     4561 ttttctaatg agggcccaaa tgtaatcacc tggctcacct tcgggtgggc ctttctgcgt
     4621 tgctg
//
