LOCUS       sequence-204422-        4023 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 BsmBI-mediated Level-3 multigene assembly..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4023)
  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 4023)
  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(−)BR
FEATURES             Location/Qualifiers
     source          1..4023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(49..222)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     171..193
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     185..202
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     186..202
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(218..234)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(218..234)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(231..253)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    242..258
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(266..272,273..290,291..296))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    311..332
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        complement(1364..1466)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1609..1626)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1780..2368)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1860..1879)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2634..2653)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2645..3460
                     /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             2645..3460
                     /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(2718..2737)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3552..4007)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3689..3710)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3901..3920
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 catggcgtct cacattgctt gcaggtgtta ttaccaggca aagcgccatt cgccattcag
       61 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
      121 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
      181 acgttgtaaa acgacggcca gtgaatccgt aatcatggtc atagctgttt cctgtgtgaa
      241 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
      301 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgccacctg cccgccgtaa
      361 gcgagagacc aagcttttat tatttaaact gtttgaggaa acgcagatcg ttttcgaaga
      421 atgaacgcag gtcggtgacg ccgtaacgca acatagtcag acgctccatc cccatcccga
      481 acccgaaacc agagtaaact tccgggtcga tgccaacgtt acgcaacacg ttcggatgca
      541 ccatcccgca gcccagcact tccagccatt taccgttttt acccatgacg tccacttctg
      601 cagaaggttc agcaaacggg aagtaggaag gacggaagcg aatctgcaaa tcttcctcaa
      661 agaagttacg caggaagtcg tgcagcgtgc ctttcaggtt ggtaaagctg atgttggtat
      721 caacaatcag accttccatc tgatggaaca tcggcgtgtg agtctggtcg tagtcgttac
      781 gataaacacg gccaggcgcg atgatacgaa tcggtggctg ctgggctttc atggtgcgga
      841 tctgtacgcc agaggtctgg gtacgcagca ggcgggtagt gtcaaaccag aaagtgtcgt
      901 ggtcagcgcg cgccgggtgg tgaccaggaa tgttcagagc atcgaagtta tgataatcgt
      961 cttcgatttc cggcccggtt gccacggtaa agccaagctc accgaagaaa ctttcgatac
     1021 ggtcgatggt acgggtaacc ggatgcagac cgccgttttc aatgcgacga cctggcagag
     1081 agacatcaat cgtttccgcc gccagacgcg cattcagtgc agcgctttcc agttccgctt
     1141 tacgcgcatt cagcgcctgc tgaacctgct ctttcgcttc gttgataacc gcaccagctg
     1201 ccggacgctc ttctggcggc agctcacgca gggtcgtcat ctgaagggtt aagtgccctt
     1261 ttttacccaa atattcgacg cgcacattat ctaacgcggc aacatctgac gcctggctaa
     1321 tggccgcctt cgcactggca accagttctg cgagatgtga cattttagct tccttagctc
     1381 ctgaaaatct cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa
     1441 agttggaacc tcttacgtgc cgatcaaagc ttggtctctc tcccgtaaga gacgctcgag
     1501 ggcatcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     1561 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     1621 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctaactcaa aggcggtaat
     1681 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     1741 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     1801 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     1861 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     1921 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     1981 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2041 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2101 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2161 gtatgtaggc ggtgctacag agttcttgaa gtggtgtcct aactacggct acactagaag
     2221 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     2281 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     2341 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     2401 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     2461 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     2521 gtaaacttgg tctgacagac cgctacgggg tctgacgctc agtggaacga aaactcacgt
     2581 taagggattt tggtcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     2641 tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat
     2701 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     2761 aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     2821 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     2881 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     2941 tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     3001 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     3061 tccttttaac agcgatcgcg tatttcgttt ggctcaggcg caatcacgaa tgaataacgg
     3121 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     3181 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     3241 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     3301 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     3361 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     3421 taaattgcag tttcatttga tgctcgatga gtttttctaa gaattaattc atgagcggat
     3481 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     3541 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     3601 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     3661 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     3721 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     3781 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     3841 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     3901 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     3961 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccagt
     4021 gtc
//
