LOCUS       sequence-201048-        5330 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Yeast endogenous mRNA tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5330)
  AUTHORS   Tutucci E, Vera M, Biswas J, Garcia J, Parker R, Singer RH
  TITLE     An improved MS2 system for accurate reporting of the mRNA life 
            cycle.
  JOURNAL   Nat Methods. 2017 Nov 13. doi: 10.1038/nmeth.4502.
  PUBMED    29131164
REFERENCE   2  (bases 1 to 5330)
  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: "Nat 
            Methods. 2017 Nov 13. doi: 10.1038/nmeth.4502."
COMMENT     Sequence Label: pET264-pUC 24xMS2V6 Loxp KANr Loxp
FEATURES             Location/Qualifiers
     source          1..5330
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     gene            6..1362
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        6..349
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             350..1159
                     /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     complement(417..436)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1165..1362
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1425..1458
                     /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(1509..1529)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1509..1527)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1548..1564)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1548..1564)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1561..1583)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1572..1588
                     /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(1596..1602,1603..1620,1621..1626))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1641..1662
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1779..1796)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1950..2538)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2030..2049)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2709..3500,3501..3569))
                     /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     3332..3351
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3570..3674)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3700..4155)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3837..3858)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4049..4068
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4282..4304
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4296..4313
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4297..4313
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4323..4342
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4323..4341
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4374..4390
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4374..4390
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    5248..5281
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 ccagcgacat ggaggcccag aataccctcc ttgacagtct tgacgtgcgc agctcagggg
       61 catgatgtga ctgtcgcccg tacatttagc ccatacatcc ccatgtataa tcatttgcat
      121 ccatacattt tgatggccgc acggcgcgaa gcaaaaatta cggctcctcg ctgcagacct
      181 gcgagcaggg aaacgctccc ctcacagacg cgttgaattg tccccacgcc gcgcccctgt
      241 agagaaatat aaaaggttag gatttgccac tgaggttctt ctttcatata cttcctttta
      301 aaatcttgct aggatacagt tctcacatca catccgaaca taaacaacca tgggtaagga
      361 aaagactcac gtttcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
      421 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa
      481 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
      541 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca
      601 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg gcaaaacagc
      661 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
      721 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt
      781 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt
      841 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataagctttt
      901 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
      961 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     1021 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     1081 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     1141 gctcgatgag tttttctaat cagtactgac aataaaaaga ttcttgtttt caagaacttg
     1201 tcatttgtat agttttttta tattgtagtt gttctatttt aatcaaatgt tagcgtgatt
     1261 tatatttttt ttcgcctcga catcatctgc ccagatgcga agttaagtgc gcagaaagta
     1321 atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac tgctgtcgat tcgatactaa
     1381 cgccgccatc cagtgtcgaa aacgagctct cgagaaccct taatataact tcgtataatg
     1441 tatgctatac gaagttatta ggtgatatca gatccactag tggcctatgc ggtacccagc
     1501 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     1561 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg gagcataaag
     1621 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     1681 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     1741 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     1801 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     1861 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     1921 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     1981 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2041 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2101 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2161 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2221 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2281 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2341 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     2401 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     2461 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2521 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     2581 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     2641 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2701 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2761 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2821 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     2881 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     2941 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3001 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3061 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3121 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3181 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3241 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3301 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3361 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3421 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3481 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3541 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     3601 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3661 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     3721 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     3781 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     3841 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     3901 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     3961 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     4021 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     4081 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     4141 cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     4201 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     4261 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg
     4321 cgtaatacga ctcactatag ggcgaattgg agctccaccg cggtggcggc cgctctagaa
     4381 ctagtggatc ccagagcccc ctggcaatcg cggggagacg caggactacc gcgtcttcac
     4441 tctcgcttgc gcggtatacg cagggagcaa cgtcacccag ggcgacacag aggaataccc
     4501 tgtgtggccc cacggtgcta caaagaacat tccgtattgc tcaccagtgt cacatgtgga
     4561 ggactacccc acaagcgagt caggaaacct tcggggcatc gcaccattat cccgaacaat
     4621 cgacacgcag gattaccgcg tgggacactc tgttccccgt caaaattgga ccataccgga
     4681 gtcggcttac gtcatgcagg attaccgcat gcatggtgca gaatatcggc atgtctactc
     4741 gtacagtcaa atctactcgt gtggtcagga ctaccgacca cttctattct attcatcttt
     4801 tcggttgtgc aggtattctg ccgatgtacg agaagacgat tacgcttctc gactaccttc
     4861 atcatacatg gtgtgcagat ggccgccaag ttttttgcca atggaggaat accccattct
     4921 ctgtcaacca acccatgcaa agtttacact ctgctatggc agcactgtcg cagaggaata
     4981 ccctgcgagc caaaacggcc cccggtgcgt gtattgcatc tgccttgcga gcattcacag
     5041 ggacgaatac gccctgccgt tgcattactt caatatgggt gctctgtcgt cgtcatcagg
     5101 accatttgcg caggactacc gcgcatatat catcagcact cgtgcggata cttctgggat
     5161 tcctattgtt acgcgagctc aggaataccg agctctggcg acagagaccc tcacacggaa
     5221 gatctatcga tctcgacaac ccttaatata acttcgtata atgtatgcta tacgaagtta
     5281 ttaggtctag agatctgttt agcttgcctc gtccccgccg ggtcacccgg
//
