LOCUS       sequence-382367-        5640 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 5640)
  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 5640)
  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: HYG-TET-OFF – ubiquitin – LEU – 4FLAG
FEATURES             Location/Qualifiers
     source          1..5640
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(70..87)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(241..829)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(321..340)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1000..1791,1792..1860))
                     /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     1623..1642
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1861..1965)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1992..2448
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2079..2098)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2289..2310
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2574..2596
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2588..2605
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2589..2605
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2615..2634
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2615..2633
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            complement(2655..4230)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(2655..2852)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(2858..3886)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(2858..3886)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     promoter        complement(3887..4230)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      4261..4448
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4316..4340)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    join(4506..4524,4525..4547,4548..4566,4567..4589,4590..4608,
                     4609..4631,4632..4650,4651..4673,4674..4692,4693..4715,
                     4716..4734,4735..4757,4758..4776)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     CDS             4984..5211
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             5230..5253
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5260..5283
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5290..5313
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5320..5343
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(5387..5403)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5387..5403)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5440..5460)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5440..5458)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5479..5495)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5479..5495)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5492..5514)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5503..5519
                     /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(5527..5533,5534..5551,5552..5557))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5572..5593
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
       61 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
      121 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
      181 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
      241 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      301 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      361 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      421 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      481 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      541 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      601 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      661 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      721 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      781 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      841 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      901 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      961 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     1021 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     1081 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     1141 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     1201 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1261 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1321 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1381 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1441 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1501 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1561 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1621 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1681 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1741 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1801 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     1861 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1921 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1981 agtgccacct gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2041 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2101 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2161 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2221 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     2281 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2341 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaaat gagctgattt
     2401 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc attcgccatt
     2461 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     2521 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     2581 acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt
     2641 gggtaccggg cccacagtat agcgaccagc attcacatac gattgacgca tgatattact
     2701 ttctgcgcac ttaacttcgc atctgggcag atgatgtcga ggcgaaaaaa aatataaatc
     2761 acgctaacat ttgattaaaa tagaacaact acaatataaa aaaactatac aaatgacaag
     2821 ttcttgaaaa caagaatctt tttattgtca gtactgatta ttcctttgcc ctcggacgag
     2881 tgctggggcg tcggtttcca ctatcggcga gtacttctac acagccatcg gtccagacgg
     2941 ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat cggacgattg
     3001 cgtcgcatcg accctgcgcc caagctgcat catcgaaatt gccgtcaacc aagctctgat
     3061 agagttggtc aagaccaatg cggagcatat acgcccggag ccgcggcgat cctgcaagct
     3121 ccggatgcct ccgctcgaag tagcgcgtct gctgctccat acaagccaac cacggcctcc
     3181 agaagaagat gttggcgacc tcgtattggg aatccccgaa catcgcctcg ctccagtcaa
     3241 tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca
     3301 cgaggtgccg gacttcgggg cagtcctcgg cccaaagcat cagctcatcg agagcctgcg
     3361 cgacggacgc actgacggtg tcgtccatca cagtttgcca gtgatacaca tggggatcag
     3421 caatcgcgca tatgaaatca cgccatgtag tgtattgacc gattccttgc ggtccgaatg
     3481 ggccgaaccc gctcgtctgg ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga
     3541 ccggctgcag aacagcgggc agttcggttt caggcaggtc ttgcaacgtg acaccctgtg
     3601 cacggcggga gatgcaatag gtcaggctct cgctgaattc cccaatgtca agcacttccg
     3661 gaatcgggag cgcggccgat gcaaagtgcc gataaacata acgatctttg tagaaaccat
     3721 cggcgcagct atttacccgc aggacatatc cgcgccctcc tacatcgaag ctgaaagcac
     3781 gagattcttc gccctccgag agctgcatca ggtcggagac gctgtcgaac ttttcgatca
     3841 gaaacttctc gacagacgtc gcggtgagtt caggcttttt acccatggtt gtttatgttc
     3901 ggatgtgatg tgagaactgt atcctagcaa gattttaaaa ggaagtatat gaaagaagaa
     3961 cctcagtggc aaatcctaac cttttatatt tctctacagg ggcgcggcgt ggggacaatt
     4021 caacgcgtct gtgaggggag cgtttccctg ctcgcaggtt cgcagcgagg agccgtaatt
     4081 tttgcttcgc gccgtgcggc catcaaaatg tatggatgca aatgattata catggggatg
     4141 tatgggctaa atgtacgggc gacagtcaca tcatgcccct gagctgccca cgtcaagact
     4201 gtcaaggagg gtattctggg cctccatgtc tctcgagtaa ttcgcgccac ttctaaataa
     4261 gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta
     4321 tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct
     4381 ttcctgtagg tcaggttgct ttctcaggta tagtatgagg tcgctcttat tgaccacacc
     4441 tctaccggca gatccgctag ggataacagg gtaatataga tcaattcctc gatcgagttt
     4501 accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga
     4561 tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc
     4621 gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat
     4681 cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg
     4741 aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gctcggtacc
     4801 ctatggcatg catgtgctct gtatgtatat aaaactcttg ttttcttctt ttctctaaat
     4861 attctttcct tatacattag gtcctttgta gcataaatta ctatacttct atagacacgc
     4921 aaacacaaat acacacacta aattaccgga tcaattcggg ggatcgacgg tatcgataag
     4981 cttatgcaaa ttttcgtcaa gactttaacc ggtaagacta ttaccctgga agttgaatct
     5041 tctgacacta ttgacaatgt caagtccaag atccaagaca aggaaggtat tccacctgac
     5101 caacaaagat tgatctttgc tggtaagcaa ttggaagatg gtagaacttt gtccgactac
     5161 aacatccaaa aggaatctac tctacacttg gtcttgagat tgagaggtgg tttgaagctt
     5221 accatggcag attacaaaga cgatgatgac aagtcaagtg actataagga tgacgatgac
     5281 aagagcagtg attataaaga tgatgacgat aaatcatctg actacaagga cgacgatgat
     5341 aaagttccag attacgctgc tcagtgcgaa ttcctgcagc ccgggggatc cactagttct
     5401 agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc
     5461 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5521 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5581 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
//
