LOCUS       sequence-244752-        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"
     primer_bind     214..236
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     228..245
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     229..245
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    246..302
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(315..331)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(315..331)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(328..350)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    339..355
                     /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(363..369,370..387,388..393))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    408..429
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(659..1247)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(739..758)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     rep_origin      1346..2505
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    1496..1543
                     /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(2547..3903)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(2547..2744)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(2750..3559)
                     /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     3473..3492
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3561..3903)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             complement(4072..4731)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4646..4665
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(join(4876..4928,1..319))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSILEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCRSLHRSS
                     NQLTGSPSRVGLSSARANC"
     terminator      complement(4894..4923)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
ORIGIN
        1 gttgattgga agacctgtgc agagacctgc accatatgcg gtgtgaaata ccgcacagat
       61 gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg
      121 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
      181 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
      241 ccagtgaatt cgagctcggt acccggggat cctctagaat cgacctgcag gcatgcaagc
      301 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
      361 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
      421 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
      481 ctgcattaat gaatcggcca acgcgcgggg gtctcacctc aggtcttcga gcggtatcag
      541 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
      601 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
      661 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
      721 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
      781 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
      841 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
      901 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
      961 atcgtcttga gcccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     1021 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     1081 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     1141 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     1201 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     1261 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     1321 tgagattatc aaaaaggatc ttcacacgca tttaagcata aacacgcact atgccgttct
     1381 tctcatgtat atatatatac aggcaacacg cagatatagg tgcgacgtga acagtgagct
     1441 gtatgtgcgc agctcgcgtt gcattttcgg aagcgctcgt tttcggaaac gctttgaagt
     1501 tcctattccg aagttcctat tctctagaaa gtataggaac ttcagagcgc ttttgaaaac
     1561 caaaagcgct ctgatgacgc actttcaaaa aaccaaaaac gcaccggact gtaacgagct
     1621 actaaaatat tgcgaatacc gcttccacaa acattgctca aaagtatctc tttgctatat
     1681 atctctgtgc tatatcccta tataacctac ccatccacct ttcgctcctt gaacttgcat
     1741 ctaaactcga cctctacatt ttttatgttt atctctagta ttactcttta gacaaaaaaa
     1801 ttgtagtaag aactattcat agagtgaatc gaaaacaata cgaaaatgta aacatttcct
     1861 atacgtagta tatagagaca aaatagaaga aaccgttcat aattttctga ccaatgaaga
     1921 atcatcaacg ctatcacttt ctgttcacaa agtatgcgca atccacatcg gtatagaata
     1981 taatcgggga tgcctttatc ttgaaaaaat gcacccgcag cttcgctagt aatcagtaaa
     2041 cgcgggaagt ggtgtcaggc tttttttatg gaagagaaaa tagacaccaa agtagccttc
     2101 ttctaacctt aacggaccta cagtgcaaaa agttatcaag agactgcatt atagagcgca
     2161 caaaggagaa aaaaagtaat ctaagatgct ttgttagaaa aatagcgctc tcgggatgca
     2221 tttttgtaga acaaaaaaga agtatagatt ctttgttggt aaaatagcgc tctcgcgttg
     2281 catttctgtt ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg
     2341 ttgcattttt gttttacaaa aatgaagcac agattcttcg ttggtaaaat agcgctttcg
     2401 cgttgcattt ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa attagcgctc
     2461 tcgcgttgca tttttgttct acaaaatgaa gcacagatgc ttcgtgaggt cgacactgga
     2521 tggcggcgtt agtatcgaat cgacagcagt atagcgacca gcattcacat acgattgacg
     2581 catgatatta ctttctgcgc acttaacttc gcatctgggc agatgatgtc gaggcgaaaa
     2641 aaaatataaa tcacgctaac atttgattaa aatagaacaa ctacaatata aaaaaactat
     2701 acaaatgaca agttcttgaa aacaagaatc tttttattgt cagtactgtt tagaaaaact
     2761 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     2821 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     2881 gatcctggta tcggtctgcg attccgacac gtccaacatc aatacaacct attaatttcc
     2941 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     3001 agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     3061 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     3121 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     3181 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     3241 cctggaatgc tgttttgccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     3301 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     3361 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     3421 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     3481 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctcgaaacgt
     3541 gtgtcttttc cttacccatt gttgtttatg ttcggatgtg atgtgagaac tgtatcctag
     3601 caagatttta aaaggaagta tatgaaagaa gaacctcagt ggcaaatcct aaccttttat
     3661 atttctctac aggggcgcgg cgtggggaca attcaacgcg tctgtgaggg gagcgtttcc
     3721 ctgctcgcag gtctgcagcg aggagccgta atttttgctt cgcgccgtgc ggccatcaaa
     3781 atgtatggat gcaaatgatt atacatgggg atgtatgggc taaatgtacg ggcgacagtc
     3841 acatcatgcc cctgagctgc gcacgtcaag actgtcaagg agggtattct gggcctccat
     3901 gtcgctggcc gggtgacccg gcggggacaa ggcaagctgc acctagatcc ttttaaatta
     3961 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagagttct
     4021 gaggtcatta ctggatctat caacagcagt ccaagcgagc tcgatatcaa attacgcccc
     4081 gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc
     4141 atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt
     4201 ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa
     4261 atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa
     4321 ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg
     4381 tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg
     4441 ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt
     4501 cattgccata cgaaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc
     4561 cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg
     4621 aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg
     4681 atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc
     4741 cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt
     4801 atggtgaaag ttggaacctc ttacgtgccc gatcaactcg cgcgtttgcc acctgacgtc
     4861 taagaaaagg aatattcagc aatttgcccg tgccgaagaa aggcccaccc gtgaaggtga
     4921 gccagtga
//
