LOCUS       sequence-252229-        4932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tagging plasmid for WTC 846: Encodes the PtetO7.1 promoter and NAT 
            resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4932)
  AUTHORS   Azizoglu A, Brent R, Rudolf F
  TITLE     A precisely adjustable, variation-suppressed eukaryotic 
            transcriptional controller to enable genetic discovery.
  JOURNAL   Elife. 2021 Aug 3;10. pii: 69549. doi: 10.7554/eLife.69549.
  PUBMED    34342575
REFERENCE   2  (bases 1 to 4932)
  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: "Elife."; 
            date: "2021-08-3"; volume: "10. pii: 69549. doi"; pages: " 
            10.7554/eLife.69549"
COMMENT     Sequence Label: FRP2375
FEATURES             Location/Qualifiers
     source          1..4932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            102..1221
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        102..445
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             446..1018
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1024..1221
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             2011..2082
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     promoter        complement(2145..2163)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2184..2200)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2184..2200)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2197..2219)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2208..2224
                     /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(2232..2238,2239..2256,2257..2262))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2277..2298
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2415..2432)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2586..3174)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2666..2685)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3345..4136,4137..4205))
                     /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     3968..3987
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4206..4310)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4336..4791)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4473..4494)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4685..4704
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(4918..4932,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4932,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg gagctcgatc
       61 ctcgagagct tgccttgtcc ccgccgggtc acccggccag cgacatggag gcccagaata
      121 ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt cgcccgtaca
      181 tttagcccat acatccccat gtataatcat ttgcatccat acattttgat ggccgcacgg
      241 cgcgaagcaa aaattacggc tcctcgctgc ggacctgcga gcagggaaac gctcccctca
      301 cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa ggttaggatt
      361 tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga tacagttctc
      421 acatcacatc cgaacataaa caaccatggg taccactctt gacgacacgg cttaccggta
      481 ccgcaccagt gtcccggggg acgccgaggc catcgaggca ctggatgggt ccttcaccac
      541 cgacaccgtc ttccgcgtca ccgccaccgg ggacggcttc accctgcggg aggtgccggt
      601 ggacccgccc ctgaccaagg tgttccccga cgacgaatcg gacgacgaat cggacgacgg
      661 ggaggacggc gacccggact cccggacgtt cgtcgcgtac ggggacgacg gcgacctggc
      721 gggcttcgtg gtcgtctcgt actccggctg gaaccgccgg ctgaccgtcg aggacatcga
      781 ggtcgccccg gagcaccggg ggcacggggt cgggcgcgcg ttgatggggc tcgcgacgga
      841 gttcgcccgc gagcggggcg ccgggcacct ctggctggag gtcaccaacg tcaacgcacc
      901 ggcgatccac gcgtaccggc ggatggggtt caccctctgc ggcctggaca ccgccctgta
      961 cgacggcacc gcctcggacg gcgagcaggc gctctacatg agcatgccct gcccctaatc
     1021 agtactgaca ataaaaagat tcttgttttc aagaacttgt catttgtata gtttttttat
     1081 attgtagttg ttctatttta atcaaatgtt agcgtgattt atattttttt tcgcctcgac
     1141 atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa tatcatgcgt caatcgtatg
     1201 tgaatgctgg tcgctatact gctgtcgatt cgatactaac gccgccatcc agtgtttaaa
     1261 cgaactagtg cggccgccag ttcgagttta tcattatcaa tactgccatt tcaaagaata
     1321 cgtaaataat taatagtagt gattttccta actttattta gtcaaaaaat tagcctctat
     1381 cattgataga gtgtctgggt gatctatcat tgatagagca tccactaatt ttaattctgc
     1441 tgtaacccgt acatgcccaa aatagggggc gggttacaca gaatctatca ttgatagagt
     1501 gtctgggtga tctatcattg atagagcatc cactaaatat aatggagctc tatcattgat
     1561 agagcatcca ctaaaaaaaa gaatcccagc accaaaatat tgttttcttc accaaccatc
     1621 agttcatagg tccattctct tagcgcaact acagagaaca ggggcacaaa caggcaaaaa
     1681 acgggcacaa cctcaatgga gtgatgcaac ctgcctggag taaatgatga cacaaggcaa
     1741 ttgacccacg catgtatcta tctcattttc ttacaccttc tattaccttc tgctctctct
     1801 gatttggaaa aagctgaaaa aaaaggttga aaccagttcc ctgaaattat tcccctatct
     1861 atcattgata gatataaata tctatcattg atagagtaat tctgtaaatc tatttcttaa
     1921 acttcttaaa ttctactttt atagttagtc ttttttttag ttttaaaaca ccaagaactt
     1981 agtttcgaat aaacacacat aaacaaaatg gattataaag atgacgatga caaggattat
     2041 aaagatgacg atgacaagga ttataaagat gacgatgaca agggaggtgg tggtggtgga
     2101 ggcggtccga cagagaatca tcgatgggta cccagctttt gttcccttta gtgagggtta
     2161 atttcgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     2221 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     2281 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     2341 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     2401 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2461 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2521 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2581 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2641 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2701 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     2761 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     2821 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     2881 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2941 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3001 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3061 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3121 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3181 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3241 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3301 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3361 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3421 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3481 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3541 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3601 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3661 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     3721 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     3781 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     3841 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3901 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg tccggcgtca
     3961 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4021 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4081 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4141 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4201 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4261 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4321 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     4381 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     4441 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     4501 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     4561 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     4621 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     4681 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     4741 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     4801 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     4861 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     4921 agtcacgacg tt
//
