LOCUS       sequence-209524-        4023 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 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(−)BF
FEATURES             Location/Qualifiers
     source          1..4023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(956..973)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1127..1715)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1207..1226)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1981..2000)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1992..2807
                     /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             1992..2807
                     /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(2065..2084)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2899..3354)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3036..3057)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3248..3267
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(3419..3592)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     3541..3563
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3555..3572
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3556..3572
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3588..3604)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3588..3604)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3601..3623)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3612..3628
                     /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(3636..3642,3643..3660,3661..3666))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3681..3702
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3747..3849
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gttatcaacg aagcgaaaga gcaggttcag caggcgctga atgcgcgtaa agcggaactg
       61 gaaagcgctg cactgaatgc gcgtctggcg gcggaaacga ttgatgtctc tctgccaggt
      121 cgtcgcattg aaaacggcgg tctgcatccg gttacccgta ccatcgaccg tatcgaaagt
      181 ttcttcggtg agcttggctt taccgtggca accgggccgg aaatcgaaga cgattatcat
      241 aacttcgatg ctctgaacat tcctggtcac cacccggcgc gcgctaacca cgacactttc
      301 tggtttgaca ctacccgcct gctgcgtacc cagacctctg gcgtacagat ccgcaccatg
      361 aaagcccagc agccaccgat tcgtatcatc gcgcctggcc gtgtttatcg taacgactac
      421 gaccagactc acacgccgat gttccatcag atggaaggtc tgattgttga taccaacatc
      481 agctttacca acctgaaagg cacgctgcac gacttcctgc gtaacttctt tgaggaagat
      541 ttgcagattc gcttccgtcc ttcctacttc ccgtttgctg aaccttctgc agaagtggac
      601 gtcatgggta aaaacggtaa atggctggaa gtgctgggct gcgggatggt gcatccgaac
      661 gtgttgcgta acgttggcat cgacccggaa gtttactctg gtttcgggtt cgggatgggg
      721 atggagcgtc tgactatgtt gcgttacggc gtcaccgacc tgcgttcatt cttcgaaaac
      781 gatctgcgtt tcctcaaaca gtttaaataa taaaagcttg gtctctcgct cgtaagagac
      841 gctcgagggc atcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
      901 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
      961 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct aactcaaagg
     1021 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     1081 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     1141 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     1201 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     1261 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     1321 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     1381 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     1441 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     1501 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtgtcctaac tacggctaca
     1561 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     1621 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     1681 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     1741 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     1801 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     1861 tatatgagta aacttggtct gacagaccgc tacggggtct gacgctcagt ggaacgaaaa
     1921 ctcacgttaa gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata
     1981 caaggggtgt tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt
     2041 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     2101 gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     2161 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     2221 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     2281 tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag
     2341 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     2401 gtaattgtcc ttttaacagc gatcgcgtat ttcgtttggc tcaggcgcaa tcacgaatga
     2461 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     2521 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     2581 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     2641 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     2701 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     2761 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg
     2821 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     2881 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     2941 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     3001 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     3061 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     3121 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     3181 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     3241 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     3301 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     3361 cgccagtgtc catggcgtct cacattgctt gcaggtgtta ttaccaggca aagcgccatt
     3421 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     3481 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     3541 cccagtcacg acgttgtaaa acgacggcca gtgaatccgt aatcatggtc atagctgttt
     3601 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3661 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgccacctg
     3721 cccgccgtag gagagagacc aagctttgat cggcacgtaa gaggttccaa ctttcaccat
     3781 aatgaaataa gatcactacc gggcgtattt tttgagttat cgagattttc aggagctaag
     3841 gaagctaaaa tgtcacatct cgcagaactg gttgccagtg cgaaggcggc cattagccag
     3901 gcgtcagatg ttgccgcgtt agataatgtg cgcgtcgaat atttgggtaa aaaagggcac
     3961 ttaacccttc agatgacgac cctgcgtgag ctgccgccag aagagcgtcc ggcagctggt
     4021 gcg
//
