LOCUS       sequence-209530-        3040 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MIDAS Level-3 destination vector. Used for initiating Level-3 
            multigene assembly..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3040)
  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 3040)
  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: pML3
FEATURES             Location/Qualifiers
     source          1..3040
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..30)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(184..772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(264..283)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             1244..2035
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWHRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      complement(2127..2582)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2264..2285)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2476..2495
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(2634..2807)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     2756..2778
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2770..2787
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2771..2787
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2803..2819)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2803..2819)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2816..2838)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2827..2843
                     /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(2851..2857,2858..2875,2876..2881))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2896..2917
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
       61 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata actcaggaaa
      121 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      181 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      241 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      301 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      361 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      421 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      481 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
      541 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
      601 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
      661 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      721 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      781 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
      841 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      901 taaatcaatc taaagtatat atgagtaaac ttggtctgac agaccggacg tcgatatccg
      961 gatgaaggca cgaacccagt ggacataagc ctgttcggtt cgtaagctgt aatgcaagta
     1021 gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa cgcagcggtg gtaacggcgc
     1081 agtggcggtt ttcatggctt gttatgactg tttttttggg gtacagtcta tgcctcgtgc
     1141 atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga gcagcaacga
     1201 tgttacgcag cagggcagtc gccctaaaac aaagttaaac atcatgaggg aagcggtgat
     1261 cgccgaagta tctactcaac tatcagaggt agttggcgtc atcgagcgcc atctcgaacc
     1321 gacgttgctg gccgtacatt tgtacggctc cgcagtggat ggcggcctga agccacacag
     1381 tgatattgat ttgctggtta cggtgaccgt aaggcttgat gaaacaacgc ggcgagcttt
     1441 gatcaacgac cttttggaaa cttcggcttc ccctggagag agcgagattc tccgcgctgt
     1501 agaagtcacc attgttgtgc acgacgacat cattccgtgg cgttatccag ctaagcgcga
     1561 actgcaattt ggagaatggc atcgcaatga cattcttgca ggtatcttcg agccagccac
     1621 gatcgacatt gatctggcta tcttgctgac aaaagcaaga gaacatagcg ttgccttggt
     1681 aggtccagcg gcggaggaac tctttgatcc ggttcctgaa caggatctat ttgaggcgct
     1741 aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg gctggcgatg agcgaaatgt
     1801 agtgcttacg ttgtcccgca tttggtacag cgcagtaacc ggcaaaatcg cgccgaagga
     1861 tgtcgctgcc gactgggcaa tggagcgcct gccggcccag tatcagcccg tcatacttga
     1921 agctagacag gcttatcttg gacaagaaga agatcgcttg gcctcgcgcg cagatcagtt
     1981 ggaagaattt gtccactacg tgaaaggcga gatcaccaag gtagtcggca aataagaatt
     2041 aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     2101 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     2161 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2221 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2281 tccactatta aagaacgtgg actccaatgt caaagggcga aaaaccgtct atcagggcga
     2341 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     2401 actaaatcgg aaccctaaag ggagcccccg atttagatct tgacggggaa agccggcgaa
     2461 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2521 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2581 gtcccattcg ccagtgtcat tgcttgcagg tgttattacc aggcaaagcg ccattcgcca
     2641 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     2701 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     2761 tcacgacgtt gtaaaacgac ggccagtgaa tccgtaatca tggtcatagc tgtttcctgt
     2821 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     2881 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgcc acctgcccgc
     2941 cgtaggcatc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3001 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
//
