LOCUS       sequence-126257-        5418 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  complementation of yeast auxotrophic marker URA3(Kluyveromyces 
            lactis).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5418)
  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 5418)
  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-Ura3Kl
FEATURES             Location/Qualifiers
     source          1..5418
                     /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"
     promoter        1167..1383
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     protein_bind    complement(2327..2360)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2425..2441)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2425..2441)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      complement(3085..3147)
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     primer_bind     complement(3230..3250)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3230..3248)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3269..3285)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3269..3285)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3282..3304)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3293..3309
                     /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(3317..3323,3324..3341,3342..3347))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3362..3383
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3500..3517)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3671..4259)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3751..3770)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4430..5221,5222..5290))
                     /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     5053..5072
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5291..5395)
                     /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 tgggtacccg
      661 ttgctaaatt cgagtgaaac acaggaagac cagaaaatcc tcatttcatc catattaaca
      721 ataatttcaa atgtttattt gcattatttg aaactaggga agacaagcaa cgaaacgttt
      781 ttgaaaattt tgagtatttt caataaattt gtagaggact cagatattga aaaaaagcta
      841 cagcaattaa tacttgataa gaagagtatt gagaagggca acggttcatc atctcatgga
      901 tctgcacatg aacaaacacc agagtcaaac gacgttgaaa ttgaggctac tgcgccaatt
      961 gatgacaata cagacgatga taacaaaccg aagttatctg atgtagaaaa ggattaaaga
     1021 tgctaagaga tagtgatgat atttcataaa taatgtaatt ctatatatgt taattacctt
     1081 ttttgcgagg catatttatg gtgaaggata agttttgacc atcaaagaag gttaatgtgg
     1141 ctgtggtttc agggtccata aagcttttca attcatcttt tttttttttg ttcttttttt
     1201 tgattccggt ttctttgaaa tttttttgat tcggtaatct ccgagcagaa ggaagaacga
     1261 aggaaggagc acagacttag attggtatat atacgcatat gtggtgttga agaaacatga
     1321 aattgcccag tattcttaac ccaactgcac agaacaaaaa cctgcaggaa acgaagataa
     1381 atcctcgaga tgtccacaaa atcatatacc agtagagctg agactcatgc aagtccggtt
     1441 gcatcgaaac ttttacgttt aatggatgaa aagaagacca atttgtgtgc ttctcttgac
     1501 gttcgttcga ctgatgagct attgaaactt gttgaaacgt tgggtccata catttgcctt
     1561 ttgaaaacac acgttgatat cttggatgat ttcagttatg agggtactgt cgttccattg
     1621 aaagcattgg cagagaaata caagttcttg atatttgagg acagaaaatt cgccgatatc
     1681 ggtaacacag tcaaattaca atatacatcg ggcgtttacc gtatcgcaga atggtctgat
     1741 atcaccaacg cccacggggt tactggtgct ggtattgttg ctggcttgaa acaaggtgcg
     1801 caagaggtca ccaaagaacc aaggggatta ttgatgcttg ctgaattgtc ttccaagggt
     1861 tctctagcac acggtgaata tactaagggt accgttgata ttgcaaagag tgataaagat
     1921 ttcgttattg ggttcattgc tcagaacgat atgggaggaa gagaagaagg gtttgattgg
     1981 ctaatcatga ccccaggtgt aggtttagac gacaaaggcg atgcattggg tcagcagtac
     2041 agaaccgtcg acgaagttgt aagtggtgga tcagatatca tcattgttgg cagaggactt
     2101 ttcgccaagg gtagagatcc taaggttgaa ggtgaaagat acagaaatgc tggatgggaa
     2161 gcgtaccaaa agagaatcag cgctccccat taattataca ggaaacttaa tagaacaaat
     2221 cacatattta atctaatagc cacctgcatt ggcacggtgc aacactactt caacttcatc
     2281 ttacaaaaag atcacgtgat ctgttgtatt gggatctcta gacctaataa cttcgtatag
     2341 catacattat acgaagttat attaagggtt gtcgacctgc agcgtacatc gataagcttg
     2401 atatcgaatt cctgcagccc gggggatcca ctagttctag agcggccgca aaaaactgta
     2461 ttataagtaa atgcatgtat actaaactca caaattagag cttcaattta attatatcag
     2521 ttattacccg ggaatctcgg tcgtaatgat ttctataatg acgaaaaaaa aaaaattgga
     2581 aagaaaaagc ttcatggcct ttataaaaag gaactatcca atacctcgcc agaaccaagt
     2641 aacagtattt tacggggcac aaatcaagaa caataagaca ggactgtaaa gatggacgca
     2701 ttgaactcca aagaacaaca agagttccaa aaagtagtgg aacaaaagca aatgaaggat
     2761 ttcatgcgtt tgtactctaa tctggtagaa agatgtttca cagactgtgt caatgacttc
     2821 acaacatcaa agctaaccaa taaggaacaa acatgcatca tgaagtgctc agaaaagttc
     2881 ttgaagcata gcgaacgtgt agggcagcgt ttccaagaac aaaacgctgc cttgggacaa
     2941 ggcttgggcc gataaggtgt actggcgtat atatatctaa ttatgtatct ctggtgtagc
     3001 ccatttttag catgtaaata taaagagaaa ccatatctaa tctaaccaaa tccaaacaaa
     3061 attcaatagt tactatcgct tttttctttc tgtatcgcaa ataagtgaaa attaaaaaag
     3121 aaagattaaa ttggaagttg gatatgggct ggaacagcag cagtaatcgg tatcgggttc
     3181 gccactaatg acgtcctacg attccgcggt ggagctccag cttttgttcc ctttagtgag
     3241 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3301 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3361 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3421 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3481 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3541 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3601 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3661 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3721 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3781 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3841 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3901 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3961 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4021 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4081 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     4141 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4201 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4261 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4321 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4381 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4441 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4501 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4561 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4621 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4681 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4741 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4801 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4861 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4921 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4981 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5041 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5101 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5161 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5221 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5281 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5341 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5401 ttccccgaaa agtgccac
//
