LOCUS       sequence-204421-        3730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MIDAS Level-2 shuttle vector for assembling transcription units 
            (TUs) with reverse 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(−)WR
FEATURES             Location/Qualifiers
     source          1..3730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1067..1169)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1316..1333)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1487..2075)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1567..1586)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2341..2360)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2352..3167
                     /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             2352..3167
                     /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(2425..2444)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3259..3714)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3396..3417)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3608..3627
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 catggcacct gcagctcatt agagacggcc ggattgcggc cgctaaccgg ccgtctcacg
       61 taagcgagag accaagcttt tattatttaa actgtttgag gaaacgcaga tcgttttcga
      121 agaatgaacg caggtcggtg acgccgtaac gcaacatagt cagacgctcc atccccatcc
      181 cgaacccgaa accagagtaa acttccgggt cgatgccaac gttacgcaac acgttcggat
      241 gcaccatccc gcagcccagc acttccagcc atttaccgtt tttacccatg acgtccactt
      301 ctgcagaagg ttcagcaaac gggaagtagg aaggacggaa gcgaatctgc aaatcttcct
      361 caaagaagtt acgcaggaag tcgtgcagcg tgcctttcag gttggtaaag ctgatgttgg
      421 tatcaacaat cagaccttcc atctgatgga acatcggcgt gtgagtctgg tcgtagtcgt
      481 tacgataaac acggccaggc gcgatgatac gaatcggtgg ctgctgggct ttcatggtgc
      541 ggatctgtac gccagaggtc tgggtacgca gcaggcgggt agtgtcaaac cagaaagtgt
      601 cgtggtcagc gcgcgccggg tggtgaccag gaatgttcag agcatcgaag ttatgataat
      661 cgtcttcgat ttccggcccg gttgccacgg taaagccaag ctcaccgaag aaactttcga
      721 tacggtcgat ggtacgggta accggatgca gaccgccgtt ttcaatgcga cgacctggca
      781 gagagacatc aatcgtttcc gccgccagac gcgcattcag tgcagcgctt tccagttccg
      841 ctttacgcgc attcagcgcc tgctgaacct gctctttcgc ttcgttgata accgcaccag
      901 ctgccggacg ctcttctggc ggcagctcac gcagggtcgt catctgaagg gttaagtgcc
      961 cttttttacc caaatattcg acgcgcacat tatctaacgc ggcaacatct gacgcctggc
     1021 taatggccgc cttcgcactg gcaaccagtt ctgcgagatg tgacatttta gcttccttag
     1081 ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt
     1141 gaaagttgga acctcttacg tgccgatcaa agcttggtct ctctcccgta atatgcaggt
     1201 gctcgagggc atcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     1261 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     1321 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct aactcaaagg
     1381 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     1441 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     1501 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     1561 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     1621 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     1681 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     1741 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     1801 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     1861 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtgtcctaac tacggctaca
     1921 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     1981 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     2041 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     2101 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     2161 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     2221 tatatgagta aacttggtct gacagaccgc tacggggtct gacgctcagt ggaacgaaaa
     2281 ctcacgttaa gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata
     2341 caaggggtgt tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt
     2401 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     2461 gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     2521 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     2581 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     2641 tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag
     2701 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     2761 gtaattgtcc ttttaacagc gatcgcgtat ttcgtttggc tcaggcgcaa tcacgaatga
     2821 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     2881 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     2941 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     3001 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     3061 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     3121 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg
     3181 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     3241 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     3301 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     3361 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     3421 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     3481 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     3541 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     3601 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     3661 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     3721 cgccagtgtc
//
