LOCUS       sequence-310755-        6323 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a shorter version of Utrophin (amino acid 35-136) with 
            amino terminus eGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6323)
  AUTHORS   Kumari A, Kesarwani S, Javoor MG, Vinothkumar KR, Sirajuddin M
  TITLE     Structural insights into actin filament recognition by commonly used
            cellular actin markers.
  JOURNAL   EMBO J. 2020 Jun 22:e104006. doi: 10.15252/embj.2019104006.
  PUBMED    32567727
REFERENCE   2  (bases 1 to 6323)
  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: "EMBO J. 
            2020 Jun 22:e104006. doi: 10.15252/embj.2019104006."
COMMENT     Sequence Label: EGFP-UTRN-mini
FEATURES             Location/Qualifiers
     source          1..6323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     17..34
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(272..291)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          274..503
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     CDS             complement(join(902..1612,1613..1615,1616..1618))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(902..1612,1613..1615,1616..1618))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(944..965)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1293..1312
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1552..1573
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1615..1624
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1685..1704)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1686..1704)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1749..1952)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1778..1798)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1953..2332
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2504..2523
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2598..2702
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2703..2771,2772..3563)
                     /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     complement(2921..2940)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3734..4322
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4223..4242
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4476..4493
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4610..4631
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4646..4651,4652..4669,4670..4676)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4684..4700
                     /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     4689..4711
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4708..4724
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4708..4724
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4784..4803)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4850..5074
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5063..5080
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(5112..5915)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5223..5242)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5833..5852
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_feature    join(5938..5946,5947..5949,5950..6323,1..201)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(6111..6130)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
ORIGIN
        1 acattcaaca gaccttgcat tcctttggcg agaggggaaa gacccctagg aatgctcgtc
       61 aagaagacag ggccaggttt ccgggccctc acattgccaa aagacggcaa tatggtggaa
      121 aataacatat agacaaacgc acaccggcct tattccaagc ggcttcggcc agtaacgtta
      181 gggggggggg agggagaggg ggggggggga gaggggcgga attggccgcc ctagatgcat
      241 gctcgacctg cagttggacc tgggagtgga cacctgtgga gagaaaggca aagtggatgt
      301 cattgtcact caagtgtatg gccagatctc aagcctgcca cacctcaagc ttgacaacaa
      361 aaagattgtc ttttctgacc agatggacgc ggccaccctc aaaggcatca ccgcgggcca
      421 ggtgaatatc aaatcctcct cgtttttgga aactgacaat cttagtgcag aagtcatgcc
      481 cgcttttgag agggagtact caccccaaca gctggccctc gcagacagcg aattaattcc
      541 agcacactgg atcagttatc tatgcggccg cttacacctg ccagtgcaaa atgatgctcc
      601 aaagtaaccc caaagtcagt ttgtgatttc catccacaat gtcggttccc cctatattca
      661 ctaattccac attgttctga tgtaaaacct gcagcactct gttgacgtta tttaaggcat
      721 gtacccttgt ggaaccacgt tcctttggca gtgatgttcc tgtgaggcct tctagaagat
      781 ccaatagctt ccttccatct ttgaggtctg tgaacatatc attgatgggt ggtttcccac
      841 tctttgaaaa tcgagcattt atccatttgg taaaggtcat cactcgagat ctgagtccgg
      901 acttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga
      961 ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt
     1021 ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg
     1081 cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt
     1141 tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt
     1201 gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca
     1261 gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga
     1321 agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct
     1381 tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg
     1441 tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt
     1501 aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt
     1561 ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatgg
     1621 tggcgaccgg tagcgctagc taggccgcag atatcgatcc gagctcggta ccaagcttgg
     1681 gtctccctat agtgagtcgt attaatttcg ataagccagt aagcagtggg ttctctagtt
     1741 agccagagag ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc
     1801 aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa
     1861 ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca
     1921 cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag
     1981 atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc
     2041 catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact
     2101 gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt
     2161 ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt
     2221 cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga
     2281 actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg tcaacgcgta
     2341 tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag attcttcatg
     2401 caattgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca ctactcagcg
     2461 acctccaaca cacaagcagg gagcagatac tggcttaact atgcggcatc agagcagatt
     2521 gtactgagag tgcaccatag gggatcggga gatctcccga tccgtcgacg tcaggtggca
     2581 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2641 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2701 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     2761 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     2821 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     2881 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     2941 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     3001 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     3061 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     3121 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     3181 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     3241 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3301 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3361 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     3421 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3481 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     3541 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3601 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3661 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3721 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3781 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3841 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     3901 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3961 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4021 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4081 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4141 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4201 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4261 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4321 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4381 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4441 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4501 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     4561 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     4621 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     4681 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     4741 tctagctaga ggtcgacggt atacagacat gataagatac attgatgagt ttggacaaac
     4801 cacaactaga atgcactcga gccccagctg gttctttccg cctcagaagc catagagccc
     4861 accgcatccc cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc
     4921 accccacccc ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt
     4981 tattaggaaa ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg
     5041 caaacaacag atggctggca actagaaggc acagtcgagg ctgatcagcg agctctagag
     5101 aattgatccc ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg
     5161 ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca
     5221 gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca
     5281 cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg
     5341 ccatgggtca cgacgagatc atcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt
     5401 tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct
     5461 tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta
     5521 gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca
     5581 ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc
     5641 cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc
     5701 cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg caccggacag gtcggtcttg
     5761 acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg
     5821 attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct
     5881 gcgtgcaatc catcttgttc aatggccgat cccatattgg ctgcaggaat tatcccgggt
     5941 tgtggcaagc ttatcatcgt gtttttcaaa ggaaaaccac gtccccgtgg ttcggggggc
     6001 ctagacgttt ttttaacctc gactaaacac atgtaaagca tgtgcaccga ggccccagat
     6061 cagatcccat acaatggggt accttctggg catccttcag ccccttgttg aatacgcttg
     6121 aggagagcca tttgactctt tccacaacta tccaactcac aacgtggcac tggggttgtg
     6181 ccgcctttgc aggtgtatct tatacacgtg gcttttggcc gcagaggcac ctgtcgccag
     6241 gtggggggtt ccgctgcctg caaagggtcg ctacagacgt tgtttgtctt caagaagctt
     6301 ccagaggaac tgcttccttc acg
//
