LOCUS       sequence-384614-        5850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gene Overexpression of Human SHARPIN S146E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5850)
  AUTHORS   Butt U, Khan MH, Pouwels J, Westermarck J
  TITLE     SHARPIN S146 phosphorylation mediates ARP2/3 interaction, cancer 
            cell invasion and metastasis.
  JOURNAL   J Cell Sci. 2022 Oct 15;135(20):jcs260627. doi: 10.1242/jcs.260627. 
            Epub 2022 Oct 13.
  PUBMED    36148554
REFERENCE   2  (bases 1 to 5850)
  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: "J Cell 
            Sci."; date: "2022-10-15"; volume: "135(20):jcs260627. doi"; pages: 
            " 10.1242/jcs.260627. Epub 2022 Oct 13"
COMMENT     Sequence Label: GFP-Sharpin S146E
FEATURES             Location/Qualifiers
     source          1..5850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /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(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2638..2759
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2675..2694)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2729..2748
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2766..3221)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2903..2924)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3115..3134
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3248..3352
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3354..3711
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3354..3545
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3377..3397)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3562..3697
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3746..4540
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3746..4540
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3800..3819)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4410..4429
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4728..4747)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4772..4819
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5148..5736
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5637..5656
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct caagcttcgg cgccgccagc gggcggggcg
     1381 gcggcggcgg cctcggactt gggctccgcc gcagtgctct tggctgtgca cgccgcggtg
     1441 aggccgctgg gcgccgggcc agacgccgag gcacagctgc ggaggctgca gctgagcgcg
     1501 gaccctgaga ggcctgggcg cttccggctg gagctgctgg gcgcgggacc tggggcggtt
     1561 aatttggagt ggcccctgga gtcagtttcc tacaccatcc gaggccccac ccagcacgag
     1621 ctacagcctc caccaggagg gcctggaacc ctcagcctgc acttcctcaa ccctcaggaa
     1681 gctcagcggt gggcagtcct agtccgaggt gccaccgtgg aaggacagaa tggcagcaag
     1741 agcaactcac caccagcctt gggcccagaa gcatgccctg tctccctgcc cgagcccccg
     1801 gaagcctcca cactcaaggg ccctccacct gaggcagatc ttcctaggag ccctggaaac
     1861 ttgacggaga gagaagagct ggcagggagc ctggcccggg ctattgcagg tggagacgag
     1921 aagggggcag cccaagtggc agccgtcctg gcccagcatc gtgtggccct gagtgttcag
     1981 cttcaggagg cctgcttccc acctggcccc atcaggttac aagtgacact tgaagacgct
     2041 gcctctgccg catccgccgc gtcctctgca cacgttgccc tgcaggtcca cccccactgc
     2101 actgttgcag ctctccagga gcaggtgttc tcagagctcg gtttcccgcc agccgtgcaa
     2161 cgctgggtca tcggacggtg cctgtgtgtg cctgagcgca gccttgcctc ttacggggtt
     2221 cggcaggatg gggaccctgc tttcctctac ttgctgtcag ctcctcgaga agccccagcc
     2281 acaggaccta gccctcagca cccccagaag atggacgggg aacttggacg cttgtttccc
     2341 ccatcattgg ggctaccccc aggcccccag ccagctgcct ccagcctgcc cagtccactc
     2401 cagcccagct ggtcctgtcc ttcctgcacc ttcatcaatg ccccagaccg ccctggctgt
     2461 gagatgtgta gcacccagag gccctgcact tgggaccccc ttgctgcagc ttccacctag
     2521 tctagataac tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     2581 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     2641 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     2701 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     2761 cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     2821 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     2881 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     2941 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     3001 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     3061 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     3121 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     3181 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     3241 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3301 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     3361 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3421 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3481 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3541 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3601 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     3661 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     3721 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3781 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3841 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     3901 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     3961 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4021 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4081 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4141 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4201 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4261 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4321 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4381 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4441 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4501 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     4561 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4621 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4681 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     4741 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     4801 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     4861 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     4921 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     4981 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5041 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5101 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5161 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5221 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5281 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5341 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5401 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5461 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5521 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5581 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5641 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5701 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5761 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5821 tctgtggata accgtattac cgccatgcat
//
