LOCUS       sequence-253011-        4928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 yeast shuttle vector for a transcription unit with A - D 
            scars. Clone in with BsaI. Has a high-copy yeast replicon and KanMX 
            selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4928)
  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 4928)
  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: pY128
FEATURES             Location/Qualifiers
     source          1..4928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(61..649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(141..160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     rep_origin      748..1907
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    898..945
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     gene            complement(1949..3305)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(1949..2146)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(2152..2961)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2875..2894
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2963..3305)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             complement(3474..4133)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4048..4067
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(4278..4649)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSILEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCRSLHRSS
                     NQLTGSPSRVGLSSARANC"
     terminator      complement(4296..4325)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     primer_bind     4544..4566
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4558..4575
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4559..4575
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    4576..4632
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(4645..4661)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4645..4661)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4658..4680)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4669..4685
                     /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(4693..4699,4700..4717,4718..4723))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4738..4759
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
       61 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      121 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      181 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      241 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      301 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      361 ctatcgtctt gagcccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      421 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      481 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      541 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      601 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      661 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      721 catgagatta tcaaaaagga tcttcacacg catttaagca taaacacgca ctatgccgtt
      781 cttctcatgt atatatatat acaggcaaca cgcagatata ggtgcgacgt gaacagtgag
      841 ctgtatgtgc gcagctcgcg ttgcattttc ggaagcgctc gttttcggaa acgctttgaa
      901 gttcctattc cgaagttcct attctctaga aagtatagga acttcagagc gcttttgaaa
      961 accaaaagcg ctctgatgac gcactttcaa aaaaccaaaa acgcaccgga ctgtaacgag
     1021 ctactaaaat attgcgaata ccgcttccac aaacattgct caaaagtatc tctttgctat
     1081 atatctctgt gctatatccc tatataacct acccatccac ctttcgctcc ttgaacttgc
     1141 atctaaactc gacctctaca ttttttatgt ttatctctag tattactctt tagacaaaaa
     1201 aattgtagta agaactattc atagagtgaa tcgaaaacaa tacgaaaatg taaacatttc
     1261 ctatacgtag tatatagaga caaaatagaa gaaaccgttc ataattttct gaccaatgaa
     1321 gaatcatcaa cgctatcact ttctgttcac aaagtatgcg caatccacat cggtatagaa
     1381 tataatcggg gatgccttta tcttgaaaaa atgcacccgc agcttcgcta gtaatcagta
     1441 aacgcgggaa gtggtgtcag gcttttttta tggaagagaa aatagacacc aaagtagcct
     1501 tcttctaacc ttaacggacc tacagtgcaa aaagttatca agagactgca ttatagagcg
     1561 cacaaaggag aaaaaaagta atctaagatg ctttgttaga aaaatagcgc tctcgggatg
     1621 catttttgta gaacaaaaaa gaagtataga ttctttgttg gtaaaatagc gctctcgcgt
     1681 tgcatttctg ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt agcgctctcg
     1741 cgttgcattt ttgttttaca aaaatgaagc acagattctt cgttggtaaa atagcgcttt
     1801 cgcgttgcat ttctgttctg taaaaatgca gctcagattc tttgtttgaa aaattagcgc
     1861 tctcgcgttg catttttgtt ctacaaaatg aagcacagat gcttcgtgag gtcgacactg
     1921 gatggcggcg ttagtatcga atcgacagca gtatagcgac cagcattcac atacgattga
     1981 cgcatgatat tactttctgc gcacttaact tcgcatctgg gcagatgatg tcgaggcgaa
     2041 aaaaaatata aatcacgcta acatttgatt aaaatagaac aactacaata taaaaaaact
     2101 atacaaatga caagttcttg aaaacaagaa tctttttatt gtcagtactg tttagaaaaa
     2161 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     2221 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     2281 aagatcctgg tatcggtctg cgattccgac acgtccaaca tcaatacaac ctattaattt
     2341 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     2401 tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     2461 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     2521 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     2581 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     2641 tacctggaat gctgttttgc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     2701 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     2761 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     2821 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     2881 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgaaac
     2941 gtgtgtcttt tccttaccca ttgttgttta tgttcggatg tgatgtgaga actgtatcct
     3001 agcaagattt taaaaggaag tatatgaaag aagaacctca gtggcaaatc ctaacctttt
     3061 atatttctct acaggggcgc ggcgtgggga caattcaacg cgtctgtgag gggagcgttt
     3121 ccctgctcgc aggtctgcag cgaggagccg taatttttgc ttcgcgccgt gcggccatca
     3181 aaatgtatgg atgcaaatga ttatacatgg ggatgtatgg gctaaatgta cgggcgacag
     3241 tcacatcatg cccctgagct gcgcacgtca agactgtcaa ggagggtatt ctgggcctcc
     3301 atgtcgctgg ccgggtgacc cggcggggac aaggcaagct gcacctagat ccttttaaat
     3361 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagagtt
     3421 ctgaggtcat tactggatct atcaacagca gtccaagcga gctcgatatc aaattacgcc
     3481 ccgccctgcc actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag
     3541 ccatcacaaa cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc
     3601 gtataatatt tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt
     3661 aaatcaaaac tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata
     3721 aaccctttag ggaaataggc caggttttca ccgtaacacg ccacatcttg cgaatatatg
     3781 tgtagaaact gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt
     3841 tgctcatgga aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct
     3901 ttcattgcca tacgaaattc cggatgagca ttcatcaggc gggcaagaat gtgaataaag
     3961 gccggataaa acttgtgctt atttttcttt acggtcttta aaaaggccgt aatatccagc
     4021 tgaacggtct ggttataggt acattgagca actgactgaa atgcctcaaa atgttcttta
     4081 cgatgccatt gggatatatc aacggtggta tatccagtga tttttttctc cattttagct
     4141 tccttagctc ctgaaaatct cgataactca aaaaatacgc ccggtagtga tcttatttca
     4201 ttatggtgaa agttggaacc tcttacgtgc ccgatcaact cgcgcgtttg ccacctgacg
     4261 tctaagaaaa ggaatattca gcaatttgcc cgtgccgaag aaaggcccac ccgtgaaggt
     4321 gagccagtga gttgattgga agacctgtgc agagacctgc accatatgcg gtgtgaaata
     4381 ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc
     4441 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     4501 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     4561 aaaacgacgg ccagtgaatt cgagctcggt acccggggat cctctagaat cgacctgcag
     4621 gcatgcaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4681 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4741 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4801 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg gtctcacctc aggtcttcga
     4861 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4921 ggaaagaa
//
