LOCUS       sequence-438457-        8103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SEMPER plasmid for expression of msfGFP[r5M] and mEBFP2 in ratios 
            dictated by the trinucleotide TIS. Relative ORF expression is 
            characterized in publication.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8103)
  AUTHORS   Duan M, Dev I, Lu A, Ayrapetyan G, You MY, Shapiro MG
  TITLE     SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic
            ribosomes for compact, ratio-tunable multi-gene expression.
  JOURNAL   Cell Syst. 2024 Jul 17;15(7):597-609.e4. doi: 
            10.1016/j.cels.2024.06.001. Epub 2024 Jul 5.
  PUBMED    38971149
REFERENCE   2  (bases 1 to 8103)
  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"; doi: 
            "10.1016/j.cels.2024.06.001"; journalName: "Cell Syst"; date: 
            "2024-07-17- 17"; volume: "15"; issue: "7"; pages: "597-609.e4"
COMMENT     Sequence Label: pCMV-SEMPER_ACC-msfGFP[r5M]_ACC-mEBFP2_IRES-mC...
FEATURES             Location/Qualifiers
     source          1..8103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     758..775
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        complement(849..953)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1021..1039
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1066..1071,1072..1089,1090..1096)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    1104..1120
                     /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)."
     primer_bind     1109..1131
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     enhancer        1150..1453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1454..1657
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1608..1628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1654..1678
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1773..1791
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1773..1790
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    3329..3880
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3473..3490)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3700..3719
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3892..4602
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4037..4055)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4305..4324
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4656..4675)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4657..4675)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5070..5658
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5123..5143)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5541..5552)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5678..6154
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5877..5896)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      6290..6744
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6377..6396)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6586..6607
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             join(7031..7099,7100..7891)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAVLGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7249..7268)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     protein_bind    7993..8026
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttattcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
      721 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
      781 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
      841 agcttgcaat tcgcgctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      901 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      961 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     1021 aataggcgta tcacgaggcc ctttcactca ttaggcaccc caggctttac actttatgct
     1081 tccggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcat
     1141 cgtgcaggtc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     1201 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     1261 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     1321 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     1381 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     1441 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     1501 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     1561 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     1621 taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc
     1681 ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct
     1741 ccggactcta gcctaggctt ttgcaaaaag ctatttaggt gacactatag aaggtacgcc
     1801 tgcaggtacc gagctcggat ccagtagttt aaaccccttc accatggtgt ccaagggcga
     1861 ggaactgttc accggcgtgg tgcccatcct ggtggaactg gacggcgacg tgaacggcca
     1921 caagttcagc gtgagaggcg agggcgaagg cgacgccaca aacggaaagc tgaccctgaa
     1981 gttcatctgc accaccggca agctgcccgt gccttggcct accctcgtga ccacactgac
     2041 ctacggcgtg cagtgcttca gcagataccc cgaccacatc aagagacacg atttcttcaa
     2101 gagcgccctg cccgagggct acgtgcagga acggaccatc agcttcaagg acgacggcac
     2161 ctacaagaca agagccgaag tgaagttcga gggcgacacc ctcgtgaacc ggatcgagct
     2221 gaagggcatc gacttcaaag aggacggcaa catcctgggc cacaagctgg agtacaactt
     2281 caacagccac aacgtgtaca tcaccgccga caagcagaaa aacggcatca aagccaactt
     2341 caagatccgg cacaacgtgg aggacggcag cgtgcagctg gccgaccact accagcagaa
     2401 cacccccatc ggagacggcc ccgtgctgct gcccgacaac cactacctga gcacacaaag
     2461 caagctgagc aaggacccca acgagaagcg ggaccacgcc gtgctgctgg aatttgtgac
     2521 cgccgctggc atcacccacg gcaaggacga gctgtacaag tgacccgggt taccatggtg
     2581 tccaagggcg aggaactgtt caccggcgtg gtgcccatcc tggtggaact ggatggcgac
     2641 gtgaacggcc acaagttctc tgtgcgggga gagggcgaag gcgacgccac aaatggcaag
     2701 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccttggcc taccctcgtg
     2761 accacactgt ctcacggcgt gcagtgcttc gccagatacc ccgaccacat gaagcagcac
     2821 gatttcttca agagcgccat gcccgagggc tacgtgcagg aacggaccat cttcttcaag
     2881 gacgacggca cctacaagac cagagccgaa gtgaagttcg agggcgacac cctcgtgaac
     2941 cggatcgagc tgaagggcgt ggacttcaaa gaggacggca acatcctggg ccacaagctg
     3001 gagtacaact tcaacagcca caacatctac atcatggccg tgaagcagaa aaacggcatc
     3061 aaagtgaact tcaagatccg gcacaacgtg gaagatggca gcgtgcagct ggccgaccac
     3121 taccagcaga acacccccat cggagatggc cccgtgctgc tgcctgatag ccactacctg
     3181 agcacccaga gcaagctgag caaggacccc aacgagaagc gggaccacat ggtgctgctg
     3241 gaatttcgga ccgccgctgg catcaccctg ggcatggatg agctgtacaa gtgataatac
     3301 taaatcgcac tgtcggcgtc cccccctaac gttactggcc gaagccgctt ggaataaggc
     3361 cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg
     3421 gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc
     3481 aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga
     3541 agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg
     3601 tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag
     3661 tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc
     3721 aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct
     3781 cggtgcacat gctttacatg tgtttagtcg aggttaaaaa acgtctaggc cccccgaacc
     3841 acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac catggtgagc
     3901 aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg
     3961 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     4021 gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg
     4081 gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac
     4141 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     4201 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc
     4261 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     4321 aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag
     4381 ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag
     4441 accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag
     4501 ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag
     4561 ggccgccact ccaccggcgg catggatgag ctgtacaagt gaaggatccc tcgaggggcc
     4621 caagcttacg cgtgcatgcg acgtcatagc tctctcccta tagtgagtcg tattataagc
     4681 tagcttggga tctttgtgaa ggaaccttac ttctgtggtg tgacataatt ggacaaacta
     4741 cctacagaga tttaaagctc taaggtaaat ataaaatttt taagtgtata atgtgttaaa
     4801 ctagctgcat atgcttgctg cttgagagtt ttgcttactg agtatgattt atgaaaatat
     4861 tatacacagg agctagtgat tctaattgtt tgtgtatttt agattcacag tcccaaggct
     4921 catttcaggc ccctcagtcc tcacagtctg ttcatgatca taatcagcca taccacattt
     4981 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     5041 atgaatgcaa ttgttgttgt tcgtggctga atcaacctct ggattacaaa atttgtgaaa
     5101 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     5161 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     5221 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     5281 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     5341 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     5401 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     5461 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     5521 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     5581 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     5641 tttgggccgc ctccccgcca gagacaatta acttcgcggg tggcatccct gtgacccctc
     5701 cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat
     5761 aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag
     5821 gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta
     5881 ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg
     5941 ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac
     6001 caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct
     6061 ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt
     6121 acaggcgtga accactgctc ccttccctgt ccttgaagtt ctcagatcct gcattaatga
     6181 atcggccaac gcgcggggag aggcggtttg cgtattggct ggcgtaatag cgaagaggcc
     6241 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     6301 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     6361 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     6421 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     6481 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     6541 tagacggttt ttcgcccttt gacgttggag tccacgttct taatagtgga ctcttgttcc
     6601 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     6661 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     6721 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     6781 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgccag gtcttggact
     6841 ggtgagaacg gcttgctcgg cagcttcgat gtgtgctgga gggagaataa aggtctaaga
     6901 tgtgcgatag agggaagtcg cattgaatta tgtgctgtgt agggatcgct ggtatcaaat
     6961 atgtgtgccc acccctggca tgagacaata accctgataa atgcttcaat aatattgaaa
     7021 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     7081 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     7141 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     7201 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     7261 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     7321 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     7381 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     7441 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     7501 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     7561 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     7621 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     7681 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     7741 gcgtgggtct cgcggtatca ttgcagtact ggggccagat ggtaagccct cccgtatcgt
     7801 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     7861 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     7921 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     7981 taatctcatg ccataacttc gtataatgta tgctatacga agttatggca tgaccaaaat
     8041 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     8101 ttc
//
