LOCUS       sequence-209244-        3730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MIDAS Level-2 shuttle vector for assembling transcription units 
            (TUs) with forward orientation and (−) polarity. TUs assembled in 
            this vector are used for AarI-mediated Level-3 multigene assembly..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3730)
  AUTHORS   van Dolleweerd CJ, Kessans SA, Van de Bittner KC, Bustamante LY, 
            Bundela R, Scott B, Nicholson MJ, Parker EJ
  TITLE     MIDAS: A Modular DNA Assembly System for Synthetic Biology.
  JOURNAL   ACS Synth Biol. 2018 Apr 20;7(4):1018-1029. doi: 
            10.1021/acssynbio.7b00363. Epub 2018 Apr 12.
  PUBMED    29620866
REFERENCE   2  (bases 1 to 3730)
  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.7b00363"; journalName: "ACS Synth Biol"; date: 
            "2018-04-20- 20"; volume: "7"; issue: "4"; pages: "1018-1029"
COMMENT     Sequence Label: pML2(−)WF
FEATURES             Location/Qualifiers
     source          1..3730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(23..40)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(194..782)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(274..293)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1048..1067)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1059..1874
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             1059..1874
                     /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
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1132..1151)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(1966..2421)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2103..2124)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2315..2334
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2517..2619
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
       61 ggcgagcggt atcagctaac tcaaaggcgg taatacggtt atccacagaa tcaggggata
      121 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
      181 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
      241 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
      301 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
      361 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
      421 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
      481 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
      541 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
      601 tgaagtggtg tcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
      661 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      721 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
      781 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
      841 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
      901 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agaccgctac
      961 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa
     1021 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa
     1081 cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     1141 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     1201 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     1261 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     1321 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc
     1381 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     1441 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     1501 gtttggctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     1561 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     1621 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     1681 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     1741 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     1801 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     1861 atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat
     1921 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     1981 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     2041 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     2101 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     2161 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     2221 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     2281 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     2341 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     2401 atgcgccgct acagggcgcg tcccattcgc cagtgtccat ggcacctgca gctcattaga
     2461 gacggccgga ttgcggccgc taaccggccg tctcacgtag gagagagacc aagctttgat
     2521 cggcacgtaa gaggttccaa ctttcaccat aatgaaataa gatcactacc gggcgtattt
     2581 tttgagttat cgagattttc aggagctaag gaagctaaaa tgtcacatct cgcagaactg
     2641 gttgccagtg cgaaggcggc cattagccag gcgtcagatg ttgccgcgtt agataatgtg
     2701 cgcgtcgaat atttgggtaa aaaagggcac ttaacccttc agatgacgac cctgcgtgag
     2761 ctgccgccag aagagcgtcc ggcagctggt gcggttatca acgaagcgaa agagcaggtt
     2821 cagcaggcgc tgaatgcgcg taaagcggaa ctggaaagcg ctgcactgaa tgcgcgtctg
     2881 gcggcggaaa cgattgatgt ctctctgcca ggtcgtcgca ttgaaaacgg cggtctgcat
     2941 ccggttaccc gtaccatcga ccgtatcgaa agtttcttcg gtgagcttgg ctttaccgtg
     3001 gcaaccgggc cggaaatcga agacgattat cataacttcg atgctctgaa cattcctggt
     3061 caccacccgg cgcgcgctga ccacgacact ttctggtttg acactacccg cctgctgcgt
     3121 acccagacct ctggcgtaca gatccgcacc atgaaagccc agcagccacc gattcgtatc
     3181 atcgcgcctg gccgtgttta tcgtaacgac tacgaccaga ctcacacgcc gatgttccat
     3241 cagatggaag gtctgattgt tgataccaac atcagcttta ccaacctgaa aggcacgctg
     3301 cacgacttcc tgcgtaactt ctttgaggaa gatttgcaga ttcgcttccg tccttcctac
     3361 ttcccgtttg ctgaaccttc tgcagaagtg gacgtcatgg gtaaaaacgg taaatggctg
     3421 gaagtgctgg gctgcgggat ggtgcatccg aacgtgttgc gtaacgttgg catcgacccg
     3481 gaagtttact ctggtttcgg gttcgggatg gggatggagc gtctgactat gttgcgttac
     3541 ggcgtcaccg acctgcgttc attcttcgaa aacgatctgc gtttcctcaa acagtttaaa
     3601 taataaaagc ttggtctctc gctcgtaata tgcaggtgct cgagggcatc actgcccgct
     3661 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3721 ggcggtttgc
//
