LOCUS       sequence-209526-        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"
     promoter        31..133
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(1174..1347)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     1296..1318
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1310..1327
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1311..1327
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1343..1359)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1343..1359)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1356..1378)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1367..1383
                     /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(1391..1397,1398..1415,1416..1421))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1436..1457
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1606..1623)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1777..2365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1857..1876)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2631..2650)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2642..3457
                     /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             2642..3457
                     /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(2715..2734)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3549..4004)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3686..3707)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3898..3917
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ggcgtctcac attggagaga gaccaagctt tgatcggcac gtaagaggtt ccaactttca
       61 ccataatgaa ataagatcac tacagggcgt attttttgag ttatcgagat tttcaggagc
      121 taaggaagct aaaatgtcac atctcgcaga actggttgcc agtgcgaagg cggccattag
      181 ccaggcgtca gatgttgccg cgttagataa tgtgcgcgtc gaatatttgg gtaaaaaagg
      241 gcacttaacc cttcagatga cgaccctgcg tgagctgccg ccagaagagc gtccggcagc
      301 tggtgcggtt atcaacgaag cgaaagagca ggttcagcag gcgctgaatg cgcgtaaagc
      361 ggaactggaa agcgctgcac tgaatgcgcg tctggcggcg gaaacgattg atgtctctct
      421 gccaggtcgt cgcattgaaa acggcggtct gcatccggtt acccgtacca tcgaccgtat
      481 cgaaagtttc ttcggtgagc ttggctttac cgtggcaacc gggccggaaa tcgaagacga
      541 ttatcataac ttcgatgctc tgaacattcc tggtcaccac ccggcgcgcg ctgaccacga
      601 cactttctgg tttgacacta cccgcctgct gcgtacccag acctctggcg tacagatccg
      661 caccatgaaa gcccagcagc caccgattcg tatcatcgcg cctggccgtg tttatcgtaa
      721 cgactacgac cagactcaca cgccgatgtt ccatcagatg gaaggtctga ttgttgatac
      781 caacatcagc tttaccaacc tgaaaggcac gctgcacgac ttcctgcgta acttctttga
      841 ggaagatttg cagattcgct tccgtccttc ctacttcccg tttgctgaac cttctgcaga
      901 agtggacgtc atgggtaaaa acggtaaatg gctggaagtg ctgggctgcg ggatggtgca
      961 tccgaacgtg ttgcgtaacg ttggcatcga cccggaagtt tactctggtt tcgggttcgg
     1021 gatggggatg gagcgtctga ctatgttgcg ttacggcgtc accgacctgc gttcattctt
     1081 cgaaaacgat ctgcgtttcc tcaaacagtt taaataataa aagcttggtc tctcgctcat
     1141 tgcttgcagg tgttattacc aggcaaagcg ccattcgcca ttcaggctgc gcaactgttg
     1201 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     1261 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     1321 ggccagtgaa tccgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     1381 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     1441 tgagctaact cacattaatt gcgttgcgcc acctgcccgc cgtaagagac gctcgagggc
     1501 atcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1561 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     1621 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct aactcaaagg cggtaatacg
     1681 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     1741 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     1801 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1861 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1921 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     1981 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2041 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2101 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2161 tgtaggcggt gctacagagt tcttgaagtg gtgtcctaac tacggctaca ctagaagaac
     2221 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2281 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2341 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2401 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2461 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2521 aacttggtct gacagaccgc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2581 gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     2641 tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga
     2701 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     2761 ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     2821 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     2881 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     2941 gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     3001 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     3061 ttttaacagc gatcgcgtat ttcgtttggc tcaggcgcaa tcacgaatga ataacggttt
     3121 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     3181 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     3241 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     3301 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     3361 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     3421 attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca
     3481 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     3541 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     3601 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     3661 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     3721 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     3781 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     3841 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     3901 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     3961 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccagtgtc
     4021 cat
//
