LOCUS       sequence-253020-        3504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 destination vector for a transcription unit, pathway 
            position 4. 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-cd
FEATURES             Location/Qualifiers
     source          1..3504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    216..249
                     /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             650..955
                     /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     834..853
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1027..1060)
                     /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(1530..1547)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1530..1546)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1645..1662)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1816..2404)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1896..1915)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2526..3335)
                     /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"
     promoter        complement(3336..3427)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(3494..3504,1..9)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 caccccttgt ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc
       61 agaagtgaaa cgccgtagcg ccgatggtag tgtggggact ccccatgcga gagtagggaa
      121 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgcccgggct
      181 aattaggggg tgtcgccctt attcgactct atagtgaagt tcctattctc tagaaagtat
      241 aggaacttct gaagtgggga cgctttccgg catcttccag accacagtat atccatccgc
      301 ctcctgttgg tgcggagacc ggcttactaa aagccagata acagtatgca tatttgcgcg
      361 ctgatttttg cggtataaga atatatactg atatgtatac ccgaagtatg tcaaaaagag
      421 gtatgctatg aagcagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa
      481 ggcatatatg atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt
      541 cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg
      601 tttattgaaa tgaacggctc ttttgctgac gagaacaggg gctggtgaaa tgcagtttaa
      661 ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat
      721 tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga
      781 taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat
      841 gaccaccgat atggccagtg tgccggtttc cgttatcggg gaagaagtgg ctgatctcag
      901 ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa tataaggtct
      961 cgcctcaacg ttgtccaggt ttgtatccac gtgtgtccgt tccgccaata ttccgcaaaa
     1021 tgaagtgaag ttcctatact ttctagagaa taggaacttc tatagtgagt cgaataaggg
     1081 cgacacaaaa tttattctaa atgcataata aatactgata acatcttata gtttgtatta
     1141 tattttgtat tatcgttgac atgtataatt ttgatatcaa aaactgattt tccctttatt
     1201 attttcgaga tttattttct taattctctt taacaaacta gaaatattgt atatacaaaa
     1261 aatcataaat aatagatgaa tagtttaatt ataggtgttc atcaatcgaa aaagcaacgt
     1321 atcttattta aagtgcgttg cttttttctc atttataagg ttaaataatt ctcatatatc
     1381 aagcaaagtg acaggcgccc ttaaatattc tgacaaatgc tctttcccta aactcccccc
     1441 ataaaaaaac ccgccgaagc gggtttttac gttatttgcg gattaacgat tactcgttat
     1501 cagaaccgcc cagggggccc gagcttaaga ctggccgtcg ttttacaaca cagaaagagt
     1561 ttgtagaaac gcaaaaaggc catccgtcag gggccttctg cttagtttga tgcctggcag
     1621 ttccctactc tcgccttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     1681 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     1741 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     1801 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     1861 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     1921 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     1981 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2041 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2101 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2161 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2221 cttgaagtgg tgggctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     2281 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2341 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     2401 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg acgcgcgcgt
     2461 aactcacgtt aagggatttt ggtcatgagc ttgcgccgtc ccgtcaagtc agcgtaatgc
     2521 tctgcttaga aaaactcatc gagcatcaaa tgaaattgca atttattcat atcaggatta
     2581 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2641 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2701 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2761 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     2821 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2881 gattgcgcct gagcgaggcg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2941 atcgagtgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     3001 ggatattctt ctaatacctg gaacgctgtt tttccgggga tcgcagtggt gagtaaccat
     3061 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gtggcataaa ttccgtcagc
     3121 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     3181 agaaacaact ctggcgcatc gggcttccca tacaagcgat agattgtcgc acctgattgc
     3241 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     3301 cgcggcctcg acgtttcccg ttgaatatgg ctcatattct tcctttttca atattattga
     3361 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3421 aaacaaatag gggtcagtgt tacaaccaat taaccaattc tgaacattat cgcgagccca
     3481 tttatacctg aatatggctc ataa
//
