LOCUS       sequence-209243-        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"
     primer_bind     complement(18..35)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(189..777)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(269..288)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1043..1062)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1054..1869
                     /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             1054..1869
                     /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(1127..1146)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(1961..2416)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2098..2119)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2310..2329
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        complement(3457..3559)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
       61 gcggtatcag ctaactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
      121 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
      181 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
      241 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
      301 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
      361 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
      421 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
      481 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
      541 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
      601 tggtgtccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
      661 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
      721 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
      781 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
      841 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
      901 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagacc gctacggggt
      961 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg
     1021 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct
     1081 tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     1141 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg
     1201 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     1261 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     1321 actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta
     1381 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     1441 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtttg
     1501 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     1561 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     1621 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     1681 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     1741 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     1801 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     1861 tttttctaag aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca
     1921 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     1981 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2041 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2101 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2161 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2221 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     2281 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     2341 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     2401 ccgctacagg gcgcgtccca ttcgccagtg tccatggcac ctgcagctca ttagcgagag
     2461 accaagcttt tattatttaa actgtttgag gaaacgcaga tcgttttcga agaatgaacg
     2521 caggtcggtg acgccgtaac gcaacatagt cagacgctcc atccccatcc cgaacccgaa
     2581 accagagtaa acttccgggt cgatgccaac gttacgcaac acgttcggat gcaccatccc
     2641 gcagcccagc acttccagcc atttaccgtt tttacccatg acgtccactt ctgcagaagg
     2701 ttcagcaaac gggaagtagg aaggacggaa gcgaatctgc aaatcttcct caaagaagtt
     2761 acgcaggaag tcgtgcagcg tgcctttcag gttggtaaag ctgatgttgg tatcaacaat
     2821 cagaccttcc atctgatgga acatcggcgt gtgagtctgg tcgtagtcgt tacgataaac
     2881 acggccaggc gcgatgatac gaatcggtgg ctgctgggct ttcatggtgc ggatctgtac
     2941 gccagaggtc tgggtacgca gcaggcgggt agtgtcaaac cagaaagtgt cgtggtcagc
     3001 gcgcgccggg tggtgaccag gaatgttcag agcatcgaag ttatgataat cgtcttcgat
     3061 ttccggcccg gttgccacgg taaagccaag ctcaccgaag aaactttcga tacggtcgat
     3121 ggtacgggta accggatgca gaccgccgtt ttcaatgcga cgacctggca gagagacatc
     3181 aatcgtttcc gccgccagac gcgcattcag tgcagcgctt tccagttccg ctttacgcgc
     3241 attcagcgcc tgctgaacct gctctttcgc ttcgttgata accgcaccag ctgccggacg
     3301 ctcttctggc ggcagctcac gcagggtcgt catctgaagg gttaagtgcc cttttttacc
     3361 caaatattcg acgcgcacat tatctaacgc ggcaacatct gacgcctggc taatggccgc
     3421 cttcgcactg gcaaccagtt ctgcgagatg tgacatttta gcttccttag ctcctgaaaa
     3481 tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga
     3541 acctcttacg tgccgatcaa agcttggtct ctctcccatt agagacggcc ggattgcggc
     3601 cgctaaccgg ccgtctcacg taatatgcag gtgctcgagg gcatcactgc ccgctttcca
     3661 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3721 tttgcgtatt
//
