LOCUS       sequence-253025-        3504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 destination vector for a transcription unit, pathway 
            position 6. Clone a transcription unit in with BsaI..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3504)
  AUTHORS   Young EM, Zhao Z, Gielesen BEM, Wu L, Benjamin Gordon D, Roubos JA, 
            Voigt CA
  TITLE     Iterative algorithm-guided design of massive strain libraries, 
            applied to itaconic acid production in yeast.
  JOURNAL   Metab Eng. 2018 Jul;48:33-43. doi: 10.1016/j.ymben.2018.05.002. Epub
            2018 May 9.
  PUBMED    29753070
REFERENCE   2  (bases 1 to 3504)
  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"; journalName: "Metab 
            Eng."; date: "2018-07"; volume: "48:33-43. doi"; pages: " 
            10.1016/j.ymben.2018.05.002. Epub 2018 May 9"
COMMENT     Sequence Label: pDSM-ef
FEATURES             Location/Qualifiers
     source          1..3504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     411..430
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     664..681
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     779..796
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     780..796
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1266..1299
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(1371..1676)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1473..1492)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2077..2110)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(2317..2336)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2403..2494
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2495..3304
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      join(3426..3504,1..510)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
       61 aatactgttc ttctagtgta gccgtagtta gcccaccact tcaagaactc tgtagcaccg
      121 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
      181 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
      241 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
      301 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
      361 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
      421 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
      481 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
      541 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
      601 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
      661 cgcagcgagt cagtgagcga ggaagcggaa ggcgagagta gggaactgcc aggcatcaaa
      721 ctaagcagaa ggcccctgac ggatggcctt tttgcgtttc tacaaactct ttctgtgttg
      781 taaaacgacg gccagtctta agctcgggcc ccctgggcgg ttctgataac gagtaatcgt
      841 taatccgcaa ataacgtaaa aacccgcttc ggcgggtttt tttatggggg gagtttaggg
      901 aaagagcatt tgtcagaata tttaagggcg cctgtcactt tgcttgatat atgagaatta
      961 tttaacctta taaatgagaa aaaagcaacg cactttaaat aagatacgtt gctttttcga
     1021 ttgatgaaca cctataatta aactattcat ctattattta tgattttttg tatatacaat
     1081 atttctagtt tgttaaagag aattaagaaa ataaatctcg aaaataataa agggaaaatc
     1141 agtttttgat atcaaaatta tacatgtcaa cgataataca aaatataata caaactataa
     1201 gatgttatca gtatttatta tgcatttaga ataaattttg tgtcgccctt attcgactca
     1261 ctatagaagt tcctattctc tagaaagtat aggaacttca cttcattttg tgcactacat
     1321 cttgtgggtg tcgacatgct ggctggattc gaaggtttcg aggcgagacc ttatattccc
     1381 cagaacatca ggttaatggc gtttttgatg tcattttcgc ggtggctgag atcagccact
     1441 tcttccccga taacggaaac cggcacactg gccatatcgg tggtcatcat gcgccagctt
     1501 tcatccccga tatgcaccac cgggtaaagt tcacgggaga ctttatctga cagcagacgt
     1561 gcactggcca gggggatcac catccgtcgc ccgggcgtgt caataatatc actctgtaca
     1621 tccacaaaca gacgataacg gctctctctt ttataggtgt aaaccttaaa ctgcatttca
     1681 ccagcccctg ttctcgtcag caaaagagcc gttcatttca ataaaccggg cgacctcagc
     1741 catcccttcc tgattttccg ctttccagcg ttcggcacgc agacgacggg cttcattctg
     1801 catggttgtg cttaccagac cggagatatt gacatcatat atgccttgag caactgatag
     1861 ctgtcgctgt caactgtcac tgtaatacgc tgcttcatag catacctctt tttgacatac
     1921 ttcgggtata catatcagta tatattctta taccgcaaaa atcagcgcgc aaatatgcat
     1981 actgttatct ggcttttagt aagccggtct ccgcacaggt acaacaagca cgaccgagca
     2041 tatgggaagg atgtttgtgg ttatttcccc acttcagaag ttcctatact ttctagagaa
     2101 taggaacttc actatagagt cgaataaggg cgacaccccc taattagccc gggcgaaagg
     2161 cccagtcttt cgactgagcc tttcgtttta tttgatgcct ggcagttccc tactctcgca
     2221 tggggagtcc ccacactacc atcggcgcta cggcgtttca cttctgagtt cggcatgggg
     2281 tcaggtggga ccaccgcgct actgccgcca ggcaaacaag gggtgttatg agccatattc
     2341 aggtataaat gggctcgcga taatgttcag aattggttaa ttggttgtaa cactgacccc
     2401 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2461 ataaatgctt caataatatt gaaaaaggaa gaatatgagc catattcaac gggaaacgtc
     2521 gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     2581 taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga
     2641 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     2701 actaaactgg ctgacggaat ttatgccact tccgaccatc aagcatttta tccgtactcc
     2761 tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcgttcc aggtattaga
     2821 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     2881 gcactcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gcctcgctca
     2941 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     3001 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     3061 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     3121 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     3181 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     3241 tggtattgat aatcctgata tgaataaatt gcaatttcat ttgatgctcg atgagttttt
     3301 ctaagcagag cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta
     3361 acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3421 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3481 ctaccagcgg tggtttgttt gccg
//
