LOCUS       sequence-197835-        6164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast endogenous mRNA tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6164)
  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 6164)
  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..6164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     238..260
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     252..269
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     253..269
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     279..298
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        279..297
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     330..346
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     330..346
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    2038..2071
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            2126..3482
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2126..2469
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2470..3279
                     /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(2537..2556)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3285..3482
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    3545..3578
                     /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(3629..3649)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3629..3647)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3668..3684)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3668..3684)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3681..3703)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3692..3708
                     /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(3716..3722,3723..3740,3741..3746))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3761..3782
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3899..3916)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4070..4658)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4150..4169)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4829..5620,5621..5689))
                     /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     5452..5471
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5690..5794)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(5821..6164,1..112)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5908..5927)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6118..6139
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
       61 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttccattcgc
      121 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
      181 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
      241 agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg
      301 aattggagct ccaccgcggt ggcggccgct ctagaactag tggatcccag agccccctgg
      361 caatcgcggg gagacgcagg actaccgcgt cttcactctc gcttgcgcgg tatacgcagg
      421 gagcaacgtc acccagggcg acacagagga ataccctgtg tggccccacg gtgctacaaa
      481 gaacattccg tattgctcac cagtgtcaca tgtggaggac taccccacaa gcgagtcagg
      541 aaaccttcgg ggcatcgcac cattatcccg aacaatcgac acgcaggatt accgcgtggg
      601 acactctgtt ccccgtcaaa attggaccat accggagtcg gcttacgtca tgcaggatta
      661 ccgcatgcat ggtgcagaat atcggcatgt ctactcgtac agtcaaatct actcgtgtgg
      721 tcaggactac cgaccacttc tattctattc atcttttcgg ttgtgcaggt attctgccga
      781 tgtacgagaa gacgattacg cttctcgact accttcatca tacatggtgt gcagatggcc
      841 gccaagtttt ttgccaatgg aggaataccc cattctctgt caaccaaccc atgcaaagtt
      901 tacactctgc tatggcagca ctgtcgcaga ggaataccct gcgagccaaa acggcccccg
      961 gtgcgtgtat tgcatctgcc ttgcgagcat tcacagggac gaatacgccc tgccgttgca
     1021 ttacttcaat atgggtgctc tgtcgtcgtc atcaggacca tttgcgcagg actaccgcgc
     1081 atatatcatc agcactcgtg cggatacttc tgggattcct attgttacgc gagctcagga
     1141 ataccgagct ctggcgacag agaccctcac acggaagatc ccagagcccc ctggcaatcg
     1201 cggggagacg caggactacc gcgtcttcac tctcgcttgc gcggtatacg cagggagcaa
     1261 cgtcacccag ggcgacacag aggaataccc tgtgtggccc cacggtgcta caaagaacat
     1321 tccgtattgc tcaccagtgt cacatgtgga ggactacccc acaagcgagt caggaaacct
     1381 tcggggcatc gcaccattat cccgaacaat cgacacgcag gattaccgcg tgggacactc
     1441 tgttccccgt caaaattgga ccataccgga gtcggcttac gtcatgcagg attaccgcat
     1501 gcatggtgca gaatatcggc atgtctactc gtacagtcaa atctactcgt gtggtcagga
     1561 ctaccgacca cttctattct attcatcttt tcggttgtgc aggtattctg ccgatgtacg
     1621 agaagacgat tacgcttctc gactaccttc atcatacatg gtgtgcagat ggccgccaag
     1681 ttttttgcca atggaggaat accccattct ctgtcaacca acccatgcaa agtttacact
     1741 ctgctatggc agcactgtcg cagaggaata ccctgcgagc caaaacggcc cccggtgcgt
     1801 gtattgcatc tgccttgcga gcattcacag ggacgaatac gccctgccgt tgcattactt
     1861 caatatgggt gctctgtcgt cgtcatcagg accatttgcg caggactacc gcgcatatat
     1921 catcagcact cgtgcggata cttctgggat tcctattgtt acgcgagctc aggaataccg
     1981 agctctggcg acagagaccc tcacacggaa gatctatcga tctcgacaac ccttaatata
     2041 acttcgtata atgtatgcta tacgaagtta ttaggtctag agatctgttt agcttgcctc
     2101 gtccccgccg ggtcacccgg ccagcgacat ggaggcccag aataccctcc ttgacagtct
     2161 tgacgtgcgc agctcagggg catgatgtga ctgtcgcccg tacatttagc ccatacatcc
     2221 ccatgtataa tcatttgcat ccatacattt tgatggccgc acggcgcgaa gcaaaaatta
     2281 cggctcctcg ctgcagacct gcgagcaggg aaacgctccc ctcacagacg cgttgaattg
     2341 tccccacgcc gcgcccctgt agagaaatat aaaaggttag gatttgccac tgaggttctt
     2401 ctttcatata cttcctttta aaatcttgct aggatacagt tctcacatca catccgaaca
     2461 taaacaacca tgggtaagga aaagactcac gtttcgaggc cgcgattaaa ttccaacatg
     2521 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     2581 atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     2641 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     2701 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     2761 gcgatccccg gcaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     2821 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     2881 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     2941 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     3001 aaagaaatgc ataagctttt gccattctca ccggattcag tcgtcactca tggtgatttc
     3061 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     3121 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     3181 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     3241 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagtactgac aataaaaaga
     3301 ttcttgtttt caagaacttg tcatttgtat agttttttta tattgtagtt gttctatttt
     3361 aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga catcatctgc ccagatgcga
     3421 agttaagtgc gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac
     3481 tgctgtcgat tcgatactaa cgccgccatc cagtgtcgaa aacgagctct cgagaaccct
     3541 taatataact tcgtataatg tatgctatac gaagttatta ggtgatatca gatccactag
     3601 tggcctatgc ggtacccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     3661 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     3721 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     3781 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3841 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3901 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3961 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4021 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4081 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4141 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4201 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4261 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4321 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4381 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4441 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4501 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4561 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4621 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4681 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4741 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4801 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4861 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4921 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4981 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5041 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5101 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5161 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5221 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5281 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5341 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5401 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5461 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5521 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5581 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5641 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5701 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5761 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5821 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5881 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5941 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     6001 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     6061 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     6121 gactcttgtt ccaaactgga acaacactca accctatctc ggtc
//
