LOCUS       sequence-383630-        5295 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Doxycycline-induced transcriptional regulation and Ubiquitin - LEU -
            4xFLAG N-terminal tagging. Ubiquitin is cleaved off, resulting in an
            unstable protein with an N-terminal leucine..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5295)
  AUTHORS   Shively CA, Dong F, Mitra RD
  TITLE     A Suite of New Strain Construction Vectors for Gene Expression 
            Knockdown in Budding Yeast.
  JOURNAL   ACS Synth Biol. 2023 Jan 17. doi: 10.1021/acssynbio.2c00547.
  PUBMED    36650116
REFERENCE   2  (bases 1 to 5295)
  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: "ACS Synth 
            Biol. 2023 Jan 17. doi: 10.1021/acssynbio.2c00547."
COMMENT     Sequence Label: KAN(G418)-TET-OFF – ubiquitin – LEU – 4FLAG
FEATURES             Location/Qualifiers
     source          1..5295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(65..82)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(236..824)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(316..335)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(995..1786,1787..1855))
                     /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     1618..1637
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1856..1960)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1987..2443
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2074..2093)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2284..2305
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2569..2591
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2583..2600
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2584..2600
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2610..2629
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2610..2628
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            complement(2650..4006)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(2650..2847)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(2853..3662)
                     /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     3576..3595
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3663..4006)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      4037..4224
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4092..4116)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    4282..4300
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4324..4342
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4366..4384
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4408..4426
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             4634..4861
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             4880..4903
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4910..4933
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4940..4963
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4970..4993
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(5037..5053)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5037..5053)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5090..5110)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5090..5108)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5129..5145)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5129..5145)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5142..5164)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5153..5169
                     /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(5177..5183,5184..5201,5202..5207))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5222..5243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
       61 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
      121 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
      181 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
      241 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
      301 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
      361 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
      421 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
      481 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
      541 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
      601 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
      661 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
      721 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
      781 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
      841 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
      901 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
      961 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     1021 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     1081 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     1141 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     1201 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     1261 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     1321 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     1381 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     1441 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     1501 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     1561 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     1621 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     1681 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     1741 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     1801 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     1861 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     1921 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     1981 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2041 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2101 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2161 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2221 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2281 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2341 atttataagg gattttgccg atttcggcct attggttaaa aaaatgagct gatttaacaa
     2401 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc
     2461 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     2521 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
     2581 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
     2641 ccgggcccac agtatagcga ccagcattca catacgattg acgcatgata ttactttctg
     2701 cgcacttaac ttcgcatctg ggcagatgat gtcgaggcga aaaaaaatat aaatcacgct
     2761 aacatttgat taaaatagaa caactacaat ataaaaaaac tatacaaatg acaagttctt
     2821 gaaaacaaga atctttttat tgtcagtact gattagaaaa actcatcgag catcaaatga
     2881 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     2941 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     3001 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     3061 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctta
     3121 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     3181 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     3241 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     3301 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttg
     3361 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     3421 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     3481 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     3541 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     3601 aaatcagcat ccatgttgga atttaatcgc ggcctcgaaa cgtgagtctt ttccttaccc
     3661 atggttgttt atgttcggat gtgatgtgag aactgtatcc tagcaagatt ttaaaaggaa
     3721 gtatatgaaa gaagaacctc agtggcaaat cctaaccttt tatatttctc tacaggggcg
     3781 cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt tccctgctcg caggtctgca
     3841 gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc aaaatgtatg gatgcaaatg
     3901 attatacatg gggatgtatg ggctaaatgt acgggcgaca gtcacatcat gcccctgagc
     3961 tgcgcacgtc aagactgtca aggagggtat tctgggcctc catgtctctc gagtaattcg
     4021 cgccacttct aaataagcga atttcttatg atttatgatt tttattatta aataagttat
     4081 aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt
     4141 attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagt atgaggtcgc
     4201 tcttattgac cacacctcta ccggcagatc cgctagggat aacagggtaa tatagatcaa
     4261 ttcctcgatc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac
     4321 cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata
     4381 gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga
     4441 gctcggtacc ctatggcatg catgtgctct gtatgtatat aaaactcttg ttttcttctt
     4501 ttctctaaat attctttcct tatacattag gtcctttgta gcataaatta ctatacttct
     4561 atagacacgc aaacacaaat acacacacta aattaccgga tcaattcggg ggatcgacgg
     4621 tatcgataag cttatgcaaa ttttcgtcaa gactttaacc ggtaagacta ttaccctgga
     4681 agttgaatct tctgacacta ttgacaatgt caagtccaag atccaagaca aggaaggtat
     4741 tccacctgac caacaaagat tgatctttgc tggtaagcaa ttggaagatg gtagaacttt
     4801 gtccgactac aacatccaaa aggaatctac tctacacttg gtcttgagat tgagaggtgg
     4861 tttgaagctt accatggcag attacaaaga cgatgatgac aagtcaagtg actataagga
     4921 tgacgatgac aagagcagtg attataaaga tgatgacgat aaatcatctg actacaagga
     4981 cgacgatgat aaagttccag attacgctgc tcagtgcgaa ttcctgcagc ccgggggatc
     5041 cactagttct agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag
     5101 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5161 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5221 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5281 acctgtcgtg ccagc
//
