LOCUS       sequence-358175-        5184 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 5184)
  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 5184)
  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: NAT-TET-OFF -LEU- 4xFLAG
FEATURES             Location/Qualifiers
     source          1..5184
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(52..69)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(223..811)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(303..322)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(982..1773,1774..1842))
                     /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     1605..1624
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1843..1947)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1974..2430
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2061..2080)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2271..2292
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2556..2578
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2570..2587
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2571..2587
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2597..2616
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2597..2615
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            complement(2637..3756)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(2637..2834)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(2840..3412)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(3413..3756)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      3787..3974
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3842..3866)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    join(4032..4050,4051..4073,4074..4092,4093..4115,4116..4134,
                     4135..4157,4158..4176,4177..4199,4200..4218,4219..4241,
                     4242..4260,4261..4283,4284..4302)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     CDS             4510..4737
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             4756..4779
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4786..4809
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4816..4839
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4846..4869
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4913..4929)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4913..4929)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4966..4986)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4966..4984)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5005..5021)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5005..5021)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5018..5040)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5029..5045
                     /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(5053..5059,5060..5077,5078..5083))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5098..5119
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
       61 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
      121 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
      181 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
      241 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
      301 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
      361 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
      421 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
      481 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
      541 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
      601 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
      661 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
      721 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
      781 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
      841 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
      901 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
      961 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     1021 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     1081 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     1141 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     1201 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     1261 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     1321 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     1381 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     1441 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     1501 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     1561 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     1621 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     1681 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     1741 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     1801 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     1861 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1921 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc
     1981 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2041 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2101 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2161 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2221 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2281 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2341 tttgccgatt tcggcctatt ggttaaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2401 attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc gcaactgttg
     2461 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     2521 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     2581 ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggcccacagt
     2641 atagcgacca gcattcacat acgattgacg catgatatta ctttctgcgc acttaacttc
     2701 gcatctgggc agatgatgtc gaggcgaaaa aaaatataaa tcacgctaac atttgattaa
     2761 aatagaacaa ctacaatata aaaaaactat acaaatgaca agttcttgaa aacaagaatc
     2821 tttttattgt cagtactgat taggggcagg gcatgctcat gtagagcgcc tgctcgccgt
     2881 ccgaggcggt gccgtcgtac agggcggtgt ccaggccgca gagggtgaac cccatccgcc
     2941 ggtacgcgtg gatcgccggt gcgttgacgt tggtgacctc cagccagagg tgcccggcac
     3001 cccgctcgcg ggcgaactcc gtcgcgagcc ccatcaacgc gcgcccgacc ccgtgccccc
     3061 ggtgctccgg ggcgacctcg atgtcctcga cggtcagccg gcggttccag ccggagtacg
     3121 agacgaccac gaagcccgcc aggtcgccgt cgtccccgta cgcgacgaac gtccgagagt
     3181 ccgggtcgcc gtcctccccg tcgtccgatt cgtcgtccga ttcgtcgtcg gggaacacct
     3241 tggtcagggg cgggtccacc ggcacctccc gcagggtgaa gccgtccccg gtggcggtga
     3301 cgcggaagac ggtgtcggtg gtgaaggacc catccagtgc ctcgatggcc tcggcgtccc
     3361 ccgggacact ggtgcggtac cggtaagccg tgtcgtcaag agtggtaccc atggttgttt
     3421 atgttcggat gtgatgtgag aactgtatcc tagcaagatt ttaaaaggaa gtatatgaaa
     3481 gaagaacctc agtggcaaat cctaaccttt tatatttctc tacaggggcg cggcgtgggg
     3541 acaattcaac gcgtctgtga ggggagcgtt tccctgctcg caggtctgca gcgaggagcc
     3601 gtaatttttg cttcgcgccg tgcggccatc aaaatgtatg gatgcaaatg attatacatg
     3661 gggatgtatg ggctaaatgt acgggcgaca gtcacatcat gcccctgagc tgcgcacgtc
     3721 aagactgtca aggagggtat tctgggcctc catgtctctc gagtaattcg cgccacttct
     3781 aaataagcga atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata
     3841 agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt
     3901 aactctttcc tgtaggtcag gttgctttct caggtatagt atgaggtcgc tcttattgac
     3961 cacacctcta ccggcagatc cgctagggat aacagggtaa tatagatcaa ttcctcgatc
     4021 gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat
     4081 cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg
     4141 aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact
     4201 ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga
     4261 aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa agtcgagctc
     4321 ggtaccctat ggcatgcatg tgctctgtat gtatataaaa ctcttgtttt cttcttttct
     4381 ctaaatattc tttccttata cattaggtcc tttgtagcat aaattactat acttctatag
     4441 acacgcaaac acaaatacac acactaaatt accggatcaa ttcgggggat cgacggtatc
     4501 gataagctta tgcaaatttt cgtcaagact ttaaccggta agactattac cctggaagtt
     4561 gaatcttctg acactattga caatgtcaag tccaagatcc aagacaagga aggtattcca
     4621 cctgaccaac aaagattgat ctttgctggt aagcaattgg aagatggtag aactttgtcc
     4681 gactacaaca tccaaaagga atctactcta cacttggtct tgagattgag aggtggtttg
     4741 aagcttacca tggcagatta caaagacgat gatgacaagt caagtgacta taaggatgac
     4801 gatgacaaga gcagtgatta taaagatgat gacgataaat catctgacta caaggacgac
     4861 gatgataaag ttccagatta cgctgctcag tgcgaattcc tgcagcccgg gggatccact
     4921 agttctagag cggccgccac cgcggtggag ctccagcttt tgttcccttt agtgagggtt
     4981 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     5041 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     5101 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5161 gtcgtgccag ctgcattaat gaat
//
