LOCUS       sequence-196884-        6947 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bicistronic expression of Utrophin-mRFP and Puro resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6947)
  TITLE     Piel Lab Plasmids
REFERENCE   2  (bases 1 to 6947)
  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"
COMMENT     Sequence Label: pIRESpuro3+mRFP-Utr_CH
FEATURES             Location/Qualifiers
     source          1..6947
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             702..1376
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             702..1376
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(832..850)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     intron          2255..2484
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2467..2486
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3006..3468
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3062..3079)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3289..3308
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3501..4100
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3501..3520)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3997..4017
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4334..4455
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4371..4390)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4556..4572)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4556..4572)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4569..4591)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4580..4596
                     /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(4604..4610,4611..4628,4629..4634))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4649..4670
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4787..4804)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4958..5546)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5038..5057)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5717..6508,6509..6577))
                     /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     6340..6359
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6578..6682)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6757..6776)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatcgat atctgcggcc tagctagcac catggcctcc tccgaggacg
      721 tcatcaagga gttcatgcgc ttcaaggtgc gcatggaggg ctccgtgaac ggccacgagt
      781 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
      841 aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtcccct cagttccagt
      901 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
      961 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
     1021 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
     1081 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcca
     1141 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagatg aggctgaagc
     1201 tgaaggacgg cggccactac gacgccgagg tcaagaccac ctacatggcc aagaagcccg
     1261 tgcagctgcc cggcgcctac aagaccgaca tcaagctgga catcacctcc cacaacgagg
     1321 actacaccat cgtggaacag tacgagcgcg ccgagggccg ccactccacc ggcgcctccg
     1381 gaaccatggc caagtatgga gaacatgaag ccagtcctga caatgggcag aacgaattca
     1441 gtgatatcat taagtccaga tctgatgaac acaatgacgt acagaagaaa acctttacca
     1501 aatggataaa tgctcgattt tcaaagagtg ggaaaccacc catcaatgat atgttcacag
     1561 acctcaaaga tggaaggaag ctattggatc ttctagaagg cctcacagga acatcactgc
     1621 caaaggaacg tggttccaca agggtacatg ccttaaataa cgtcaacaga gtgctgcagg
     1681 ttttacatca gaacaatgtg gaattagtga atataggggg aaccgacatt gtggatggaa
     1741 atcacaaact gactttgggg ttactttgga gcatcatttt gcactggcag gtgaaagatg
     1801 tcatgaagga tgtcatgtcg gacctgcagc agacgaacag tgagaagatc ctgctcagct
     1861 gggtgcgtca gaccaccagg ccctacagcc aagtcaacgt cctcaacttc accaccagct
     1921 ggacagatgg actcgccttt aatgctgtcc tccaccgaca taaacctgat ctcttcagct
     1981 gggataaagt tgtcaaaatg tcaccaattg agagacttga acatgccttc agcaaggctc
     2041 aaacttattt gggaattgaa aagctgttag atcctgaaga tgttgccgtt cggcttcctg
     2101 acaagaaatc cataattatg tatttaacat ctttgtttga ggtgctacct cagcaagtca
     2161 ccatagacta attcgaattc ggatccgcgg ccgcatagat aactgatcca gtgtgctgga
     2221 attaattcgc tgtctgcgag ggccagctgt tggggtgagt actccctctc aaaagcgggc
     2281 atgacttctg cgctaagatt gtcagtttcc aaaaacgagg aggatttgat attcacctgg
     2341 cccgcggtga tgcctttgag ggtggccgcg tccatctggt cagaaaagac aatctttttg
     2401 ttgtcaagct tgaggtgtgg caggcttgag atctggccat acacttgagt gacaatgaca
     2461 tccactttgc ctttctctcc acaggtgtcc actcccaggt ccaactgcag gtcgagcatg
     2521 catctagggc ggccaattcc gcccctctcc cccccgttca tctacaaggt gaagctgcgc
     2581 ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
     2641 tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg
     2701 aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag
     2761 cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac
     2821 gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc
     2881 atggacgagc tgtacaagta aggcgcgccc ccccctaacg ttactggccg aagccgcttg
     2941 gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc
     3001 aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc
     3061 cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa
     3121 gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct
     3181 ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca
     3241 caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca
     3301 agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat
     3361 ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc
     3421 cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataagc ttgccacaac
     3481 ccacaaggag acgaccttcc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     3541 acgacgtccc ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     3601 gccacaccgt cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     3661 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     3721 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     3781 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     3841 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc
     3901 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     3961 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     4021 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     4081 cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg
     4141 cacgacccca tggctccgac cgaagccacc cggggcggcc ccgccgaccc cgcacccgcc
     4201 cccgaggccc accgactcta gataactgat cataatcagc cataccacat ttgtagaggt
     4261 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     4321 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4381 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4441 catcaatgta tcttaacgct cgagtgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4501 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     4561 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4621 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4681 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4741 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4801 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4861 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4921 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4981 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5041 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5101 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5161 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5221 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5281 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5341 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5401 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5461 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5521 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5581 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5641 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5701 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5761 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5821 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5881 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5941 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6001 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6061 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6121 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6181 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6241 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6301 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6361 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6421 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6481 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6541 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6601 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6661 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     6721 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     6781 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     6841 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     6901 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgtt
//
