LOCUS       sequence-209534-        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        153..255
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      1338..1793
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1425..1444)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1635..1656
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1885..2700)
                     /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             complement(1885..2700)
                     /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     2608..2627
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2692..2711
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2977..3565
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3466..3485
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(3719..3730,1..6)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
       61 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga tgccctcgag
      121 cacctgcata ttacgggaga gagaccaagc tttgatcggc acgtaagagg ttccaacttt
      181 caccataatg aaataagatc actaccgggc gtattttttg agttatcgag attttcagga
      241 gctaaggaag ctaaaatgtc acatctcgca gaactggttg ccagtgcgaa ggcggccatt
      301 agccaggcgt cagatgttgc cgcgttagat aatgtgcgcg tcgaatattt gggtaaaaaa
      361 gggcacttaa cccttcagat gacgaccctg cgtgagctgc cgccagaaga gcgtccggca
      421 gctggtgcgg ttatcaacga agcgaaagag caggttcagc aggcgctgaa tgcgcgtaaa
      481 gcggaactgg aaagcgctgc actgaatgcg cgtctggcgg cggaaacgat tgatgtctct
      541 ctgccaggtc gtcgcattga aaacggcggt ctgcatccgg ttacccgtac catcgaccgt
      601 atcgaaagtt tcttcggtga gcttggcttt accgtggcaa ccgggccgga aatcgaagac
      661 gattatcata acttcgatgc tctgaacatt cctggtcacc acccggcgcg cgctgaccac
      721 gacactttct ggtttgacac tacccgcctg ctgcgtaccc agacctctgg cgtacagatc
      781 cgcaccatga aagcccagca gccaccgatt cgtatcatcg cgcctggccg tgtttatcgt
      841 aacgactacg accagactca cacgccgatg ttccatcaga tggaaggtct gattgttgat
      901 accaacatca gctttaccaa cctgaaaggc acgctgcacg acttcctgcg taacttcttt
      961 gaggaagatt tgcagattcg cttccgtcct tcctacttcc cgtttgctga accttctgca
     1021 gaagtggacg tcatgggtaa aaacggtaaa tggctggaag tgctgggctg cgggatggtg
     1081 catccgaacg tgttgcgtaa cgttggcatc gacccggaag tttactctgg tttcgggttc
     1141 gggatgggga tggagcgtct gactatgttg cgttacggcg tcaccgacct gcgttcattc
     1201 ttcgaaaacg atctgcgttt cctcaaacag tttaaataat aaaagcttgg tctctcgctt
     1261 acgtgagacg gccggttagc ggccgcaatc cggccgtctc taatgagctg caggtgccat
     1321 ggacactggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1381 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1441 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     1501 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     1561 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     1621 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     1681 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     1741 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     1801 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     1861 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     1921 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     1981 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     2041 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     2101 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     2161 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     2221 caaaccgtta ttcattcgtg attgcgcctg agccaaacga aatacgcgat cgctgttaaa
     2281 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     2341 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     2401 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     2461 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     2521 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     2581 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     2641 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     2701 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     2761 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agcggtctgt cagaccaagt
     2821 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2881 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2941 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3001 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3061 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3121 tgttcttcta gtgtagccgt agttaggaca ccacttcaag aactctgtag caccgcctac
     3181 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3241 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3301 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3361 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3421 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3481 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3541 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3601 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3661 ccgtattacc gcctttgagt tagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3721 cgagtcagtg
//
