LOCUS       sequence-209245-        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"
     protein_bind    887..908
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(923..928,929..946,947..953)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    961..977
                     /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)."
     primer_bind     966..988
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     985..1001
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     985..1001
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             997..1170
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     complement(1017..1034)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1017..1033)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1026..1048)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1235..1690
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1322..1341)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1532..1553
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1782..2597)
                     /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             complement(1782..2597)
                     /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     2505..2524
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2589..2608
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2874..3462
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3363..3382
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3616..3633
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3776..3878
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 cgccagaaga gcgtccggca gctggtgcgg ttatcaacga agcgaaagag caggttaagc
       61 aggcgctgaa tgcgcgtaaa gcggaactgg aaagcgctgc actgaatgcg cgtctggcgg
      121 cggaaacgat tgatgtctct ctgccaggtc gtcgcattga aaacggcggt ctgcatccgg
      181 ttacccgtac catcgaccgt atcgaaagtt tcttcggtga gcttggcttt accgtggcaa
      241 ccgggccgga aatcgaagac gattatcata acttcgatgc tctgaacatt cctggtcacc
      301 acccggcgcg cgctgaccac gacactttct ggtttgacac tacccgcctg ctgcgtaccc
      361 agacctctgg cgtacagatc cgcaccatga aagcccagca gccaccgatt cgtatcatcg
      421 cgcctggccg tgtttatcgt aacgactacg accagactca cacgccgatg ttccatcaga
      481 tggaaggtct gattgttgat accaacatca gctttaccaa cctgaaaggc acgctgcacg
      541 acttcctgcg taacttcttt gaggaagatt tgcagattcg cttccgtcct tcctacttcc
      601 cgtttgctga accttctgca gaagtggacg tcatgggtaa aaacggtaaa tggctggaag
      661 tgctgggctg cgggatggtg catccgaacg tgttgcgtaa cgttggcatc gacccggaag
      721 tttactctgg tttcgggttc gggatgggga tggagcgtct gactatgttg cgttacggcg
      781 tcaccgacct gcgttcattc ttcgaaaacg atctgcgttt cctcaaacag tttaaataat
      841 aaaagcttgg tctctcgctt acggcgggca ggtggcgcaa cgcaattaat gtgagttagc
      901 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      961 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac ggattcactg
     1021 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     1081 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     1141 tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg cctggtaata acacctgcaa
     1201 gcaatgtgag acgccatgga cactggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1261 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1321 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1381 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1441 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1501 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1561 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1621 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1681 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1741 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     1801 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     1861 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     1921 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     1981 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2041 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2101 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc caaacgaaat
     2161 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2221 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2281 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2341 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2401 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2461 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2521 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     2581 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     2641 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtagc
     2701 ggtctgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     2761 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     2821 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2881 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2941 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3001 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggacacca cttcaagaac
     3061 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3121 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3181 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3241 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3301 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3361 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3421 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3481 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3541 cctgattctg tggataaccg tattaccgcc tttgagttag ctgataccgc tcgccgcagc
     3601 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     3661 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     3721 tggaaagcgg gcagtgatgc cctcgagcgt ctcttacggg agagagacca agctttgatc
     3781 ggcacgtaag aggttccaac tttcaccata atgaaataag atcactaccg ggcgtatttt
     3841 ttgagttatc gagattttca ggagctaagg aagctaaaat gtcacatctc gcagaactgg
     3901 ttgccagtgc gaaggcggcc attagccagg cgtcagatgt tgccgcgtta gataatgtgc
     3961 gcgtcgaata tttgggtaaa aaagggcact taacccttca gatgacgacc ctgcgtgagc
     4021 tgc
//
