LOCUS       sequence-214578-        4022 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 4022)
  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 4022)
  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..4022
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1037..1138)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(1192..1365)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     1314..1336
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1328..1345
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1329..1345
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1361..1377)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1361..1377)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1374..1396)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1385..1401
                     /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(1409..1415,1416..1433,1434..1439))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1454..1475
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1624..1641)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1795..2383)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1875..1894)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2649..2668)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2660..3475
                     /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             2660..3475
                     /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(2733..2752)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3567..4022)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3704..3725)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3916..3935
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cccattcgcc agtgtccatg gcgtctcaca ttagcgagag accaagcttt tattatttaa
       61 actgtttgag gaaacgcaga tcgttttcga agaatgaacg caggtcggtg acgccgtaac
      121 gcaacatagt cagacgctcc atccccatcc cgaacccgaa accagagtaa acttccgggt
      181 cgatgccaac gttacgcaac acgttcggat gcaccatccc gcagcccagc acttccagcc
      241 atttaccgtt tttacccatg acgtccactt ctgcagaagg ttcagcaaac gggaagtagg
      301 aaggacggaa gcgaatctgc aaatcttcct caaagaagtt acgcaggaag tcgtgcagcg
      361 tgcctttcag gttggtaaag ctgatgttgg tatcaacaat cagaccttcc atctgatgga
      421 acatcggcgt gtgagtctgg tcgtagtcgt tacgataaac acggccaggc gcgatgatac
      481 gaatcggtgg ctgctgggct ttcatggtgc ggatctgtac gccagaggtc tgggtacgca
      541 gcaggcgggt agtgtcaaac cagaaagtgt cgtggtcagc gcgcgccggg tggtgaccag
      601 gaatgttcag agcatcgaag ttatgataat cgtcttcgat ttccggcccg gttgccacgg
      661 taaagccaag ctcaccgaag aaactttcga tacggtcgat ggtacgggta accggatgca
      721 gaccgccgtt ttcaatgcga cgacctggca gagagacatc aatcgtttcc gccgccagac
      781 gcgcattcag tgcagcgctt tccagttccg ctttacgcgc attcagcgcc tgctgaacct
      841 gctctttcgc ttcgttgata accgcaccag ctgccggacg ctcttctggc ggcagctcac
      901 gcagggtcgt catctgaagg gttaagtgcc cttttttacc caaatattcg acgcgcacat
      961 tatctaacgc ggcaacatct gacgcctggc taatggccgc cttcgcactg gcaaccagtt
     1021 ctgcgagatg tgacatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata
     1081 cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttagt gccgatcaaa
     1141 gcttggtctc tctcccattg cttgcaggtg ttattaccag gcaaagcgcc attcgccatt
     1201 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     1261 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     1321 acgacgttgt aaaacgacgg ccagtgaatc cgtaatcatg gtcatagctg tttcctgtgt
     1381 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     1441 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgccac ctgcccgccg
     1501 taagagacgc tcgagggcat cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     1561 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     1621 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctaa
     1681 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     1741 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     1801 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     1861 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1921 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     1981 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2041 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2101 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2161 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt gtcctaacta
     2221 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2281 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     2341 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     2401 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     2461 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2521 ctaaagtata tatgagtaaa cttggtctga cagaccgcta cggggtctga cgctcagtgg
     2581 aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa
     2641 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg
     2701 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     2761 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
     2821 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     2881 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     2941 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
     3001 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     3061 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtttggctc aggcgcaatc
     3121 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     3181 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     3241 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     3301 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     3361 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     3421 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt
     3481 aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3541 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     3601 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     3661 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     3721 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     3781 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     3841 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     3901 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     3961 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     4021 gt
//
