LOCUS       sequence-128238-        5155 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  complementation of yeast auxotrophic marker LEU2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5155)
  AUTHORS   Li D, Song JZ, Shan MH, Li SP, Liu W, Li H, Zhu J, Wang Y, Lin J, 
            Xie Z
  TITLE     A fluorescent tool set for yeast Atg proteins.
  JOURNAL   Autophagy. 2015;11(6):954-60. doi: 10.1080/15548627.2015.1040971.
  PUBMED    25998947
REFERENCE   2  (bases 1 to 5155)
  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: 
            "Autophagy."; date: "2015"; volume: "11(6):954-60. doi"; pages: " 
            10.1080/15548627.2015.1040971"
COMMENT     Sequence Label: BS-Leu2
FEATURES             Location/Qualifiers
     source          1..5155
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1031..2125
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(2914..2930)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2914..2930)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2967..2987)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2967..2985)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3006..3022)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3006..3022)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3019..3041)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3030..3046
                     /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(3054..3060,3061..3078,3079..3084))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3099..3120
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3237..3254)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3408..3996)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3488..3507)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4167..4958,4959..5027))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4790..4809
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5028..5132)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccga
      661 gttcgaatct cttagcaacc attatttttt tcctcaacat aacgagaaca cacaggggcg
      721 ctatcgcaca gaatcaaatt cgatgactgg aaattttttg ttaatttcag aggtcgcctg
      781 acgcatatac ctttttcaac tgaaaaattg ggagaaaaag gaaaggtgag agcgccggaa
      841 ccggcttttc atatagaata gagaagcgtt catgactaaa tgcttgcatc acaatacttg
      901 aagttgacaa tattatttaa ggacctattg ttttttccaa taggtggtta gcaatcgtct
      961 tactttctaa cttttcttac cttttacatt tcagcaatat atatatatat ttcaaggata
     1021 taccattcta atgtctgccc ctaagaagat cgtcgttttg ccaggtgacc acgttggtca
     1081 agaaatcaca gccgaagcca ttaaggttct taaagctatt tctgatgttc gttccaatgt
     1141 caagttcgat ttcgaaaatc atttaattgg tggtgctgct atcgatgcta caggtgttcc
     1201 acttccagat gaggcgctgg aagcctccaa gaaggctgat gccgttttgt taggtgctgt
     1261 gggtggtcct aaatggggta ccggtagtgt tagacctgaa caaggtttac taaaaatccg
     1321 taaagaactt caattgtacg ccaacttaag accatgtaac tttgcatccg actctctttt
     1381 agacttatct ccaatcaagc cacaatttgc taaaggtact gacttcgttg ttgtcagaga
     1441 attagtggga ggtatttact ttggtaagag aaaggaagac gatggtgatg gtgtcgcttg
     1501 ggatagtgaa caatacaccg ttccagaagt gcaaagaatc acaagaatgg ccgctttcat
     1561 ggccctacaa catgagccac cattgcctat ttggtccttg gataaagcta atgttttggc
     1621 ctcttcaaga ttatggagaa aaactgtgga ggaaaccatc aagaacgaat tccctacatt
     1681 gaaggttcaa catcaattga ttgattctgc cgccatgatc ctagttaaga acccaaccca
     1741 cctaaatggt attataatca ccagcaacat gtttggtgat atcatctccg atgaagcctc
     1801 cgttatccca ggttccttgg gtttgttgcc atctgcgtcc ttggcctctt tgccagacaa
     1861 gaacaccgca tttggtttgt acgaaccatg ccacggttct gctccagatt tgccaaagaa
     1921 taaggttgac cctatcgcca ctatcttgtc tgctgcaatg atgttgaaat tgtcattgaa
     1981 cttgcctgaa gaaggtaagg ccattgaaga tgcagttaaa aaggttttgg atgcaggtat
     2041 cagaactggt gatttaggtg gttccaacag taccaccgaa gtcggtgatg ctgtcgccga
     2101 agaagttaag aaaatccttg cttaactcga ggaatatgtt catagggtag acgaaactat
     2161 atacgcaatc tacatacatt tatcaagaag gagaaaaagg aggatgtaaa ggaatacagg
     2221 taagcaaatt gatactaatg gctcaacgtg ataaggaaaa agaattgcac tttaacatta
     2281 atattgacaa ggaggagggc accacacaaa aagttaggtg taacagaaaa tcatgaaact
     2341 atgattccta atttatatat tggaggattt tctctaaaaa aaaaaaaata caacaaataa
     2401 aaaacactca atgacctgac catttgatgg agtttaagtc aataccttct tgaaccattt
     2461 cccataatgg tgaaagttcc ctcaagaatt ttactctgtc agaaacggcc ttaacgacgt
     2521 agtcgacctc ctcttcagta ctaaatctac caataccaaa tctgatggaa gaatgggcta
     2581 atgcatcatc cttacccagc gcatgtaaaa cataagaagg ttctagggaa gcagatgtac
     2641 aggctgaacc cgaggataat gcgatatccc ttagtgccat caataaagat tctccttcca
     2701 cgtaggcgaa agaaacgtta acacaccctg gataacgatg atctggagat ccgttcaacg
     2761 tggtatgttc agcggataat agacctttga ctaatttatc ggatagtctt ttgatgtgag
     2821 cttggtcgtt gtcaaattct ttcttcatca atctcgcagc ttcaccaaat cccgctacca
     2881 ataagcttga tatcgaattc ctgcagcccg ggggatccac tagttctaga gcggccgcca
     2941 ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     3001 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3061 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3121 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3181 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3241 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3301 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3361 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3421 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3481 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3541 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3601 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3661 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3721 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3781 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3841 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3901 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3961 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4021 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4081 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4141 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4201 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4261 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4321 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4381 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4441 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4501 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4561 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4621 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4681 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4741 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4801 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4861 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4921 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4981 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5041 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5101 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccac
//
