LOCUS       sequence-209522-        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"
     promoter        114..216
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1392..1409)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1563..2151)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1643..1662)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2417..2436)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2428..3243
                     /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             2428..3243
                     /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(2501..2520)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(3335..3730,1..60))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3472..3493)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3684..3703
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
       61 cccattcgcc agtgtccatg gcacctgcag ctcattggag agagaccaag ctttgatcgg
      121 cacgtaagag gttccaactt tcaccataat gaaataagat cactaccggg cgtatttttt
      181 gagttatcga gattttcagg agctaaggaa gctaaaatgt cacatctcgc agaactggtt
      241 gccagtgcga aggcggccat tagccaggcg tcagatgttg ccgcgttaga taatgtgcgc
      301 gtcgaatatt tgggtaaaaa agggcactta acccttcaga tgacgaccct gcgtgagctg
      361 ccgccagaag agcgtccggc agctggtgcg gttatcaacg aagcgaaaga gcaggttcag
      421 caggcgctga atgcgcgtaa agcggaactg gaaagcgctg cactgaatgc gcgtctggcg
      481 gcggaaacga ttgatgtctc tctgccaggt cgtcgcattg aaaacggcgg tctgcatccg
      541 gttacccgta ccatcgaccg tatcgaaagt ttcttcggtg agcttggctt taccgtggca
      601 accgggccgg aaatcgaaga cgattatcat aacttcgatg ctctgaacat tcctggtcac
      661 cacccggcgc gcgctgacca cgacactttc tggtttgaca ctacccgcct gctgcgtacc
      721 cagacctctg gcgtacagat ccgcaccatg aaagcccagc agccaccgat tcgtatcatc
      781 gcgcctggcc gtgtttatcg taacgactac gaccagactc acacgccgat gttccatcag
      841 atggaaggtc tgattgttga taccaacatc agctttacca acctgaaagg cacgctgcac
      901 gacttcctgc gtaacttctt tgaggaagat ttgcagattc gcttccgtcc ttcctacttc
      961 ccgtttgctg aaccttctgc agaagtggac gtcatgggta aaaacggtaa atggctggaa
     1021 gtgctgggct gcgggatggt gcatccgaac gtgttgcgta acgttggcat cgacccggaa
     1081 gtttactctg gtttcgggtt cgggatgggg atggagcgtc tgactatgtt gcgttacggc
     1141 gtcaccgacc tgcgttcatt cttcgaaaac gatctgcgtt tcctcaaaca gtttaaataa
     1201 taaaagcttg gtctctcgct cattagagac ggccggattg cggccgctaa ccggccgtct
     1261 cacgtaatat gcaggtgctc gagggcatca ctgcccgctt tccagtcggg aaacctgtcg
     1321 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1381 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1441 tcagctaact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1501 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1561 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1621 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1681 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1741 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1801 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1861 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     1921 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgt
     1981 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2041 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2101 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2161 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2221 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2281 aaatcaatct aaagtatata tgagtaaact tggtctgaca gaccgctacg gggtctgacg
     2341 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct
     2401 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct
     2461 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     2521 aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag
     2581 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     2641 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     2701 gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa
     2761 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     2821 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tttggctcag
     2881 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     2941 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     3001 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     3061 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     3121 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     3181 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     3241 taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     3301 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta
     3361 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     3421 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     3481 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     3541 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     3601 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     3661 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     3721 gcaagtgtag
//
