LOCUS       sequence-197834-        5330 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 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: pET251-pUC 12xMS2V6 Loxp KANr Loxp
FEATURES             Location/Qualifiers
     source          1..5330
                     /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    1204..1237
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            1292..2648
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1292..1635
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1636..2445
                     /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(1703..1722)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2451..2648
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    2711..2744
                     /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(2795..2815)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2795..2813)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2834..2850)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2834..2850)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2847..2869)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2858..2874
                     /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(2882..2888,2889..2906,2907..2912))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2927..2948
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3065..3082)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3236..3824)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3316..3335)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3995..4786,4787..4855))
                     /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     4618..4637
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4856..4960)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(4987..5330,1..112)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5074..5093)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5284..5305
                     /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 tatcgatctc gacaaccctt
     1201 aatataactt cgtataatgt atgctatacg aagttattag gtctagagat ctgtttagct
     1261 tgcctcgtcc ccgccgggtc acccggccag cgacatggag gcccagaata ccctccttga
     1321 cagtcttgac gtgcgcagct caggggcatg atgtgactgt cgcccgtaca tttagcccat
     1381 acatccccat gtataatcat ttgcatccat acattttgat ggccgcacgg cgcgaagcaa
     1441 aaattacggc tcctcgctgc agacctgcga gcagggaaac gctcccctca cagacgcgtt
     1501 gaattgtccc cacgccgcgc ccctgtagag aaatataaaa ggttaggatt tgccactgag
     1561 gttcttcttt catatacttc cttttaaaat cttgctagga tacagttctc acatcacatc
     1621 cgaacataaa caaccatggg taaggaaaag actcacgttt cgaggccgcg attaaattcc
     1681 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     1741 gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     1801 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     1861 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     1921 accactgcga tccccggcaa aacagcattc caggtattag aagaatatcc tgattcaggt
     1981 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     2041 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     2101 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     2161 gtctggaaag aaatgcataa gcttttgcca ttctcaccgg attcagtcgt cactcatggt
     2221 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     2281 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     2341 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     2401 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcagt actgacaata
     2461 aaaagattct tgttttcaag aacttgtcat ttgtatagtt tttttatatt gtagttgttc
     2521 tattttaatc aaatgttagc gtgatttata ttttttttcg cctcgacatc atctgcccag
     2581 atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga atgctggtcg
     2641 ctatactgct gtcgattcga tactaacgcc gccatccagt gtcgaaaacg agctctcgag
     2701 aacccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtg atatcagatc
     2761 cactagtggc ctatgcggta cccagctttt gttcccttta gtgagggtta attgcgcgct
     2821 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2881 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2941 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3001 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3061 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3121 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3181 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3241 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3301 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3361 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3421 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3481 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3541 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3601 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3661 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3721 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3781 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3841 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3901 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3961 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4021 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4081 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4141 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4201 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4261 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4321 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4381 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4441 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4501 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4561 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4621 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4681 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4741 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4801 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4861 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4921 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4981 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     5041 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     5101 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5161 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     5221 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5281 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
//
