LOCUS       sequence-174179-        6290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP-tagged mSin1 (isoform 1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6290)
  AUTHORS   Ebner M, Sinkovics B, Szczygiel M, Ribeiro DW, Yudushkin I
  TITLE     Localization of mTORC2 activity inside cells.
  JOURNAL   J Cell Biol. 2017 Jan 31. pii: jcb.201610060. doi: 
            10.1083/jcb.201610060.
  PUBMED    28143890
REFERENCE   2  (bases 1 to 6290)
  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 
            Biol. 2017 Jan 31. pii: jcb.201610060. doi: 10.1083/jcb.201610060."
COMMENT     Sequence Label: N1-mSin1.1-GFP
FEATURES             Location/Qualifiers
     source          1..6290
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(6..26)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      191..326
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             375..1169
                     /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             375..1169
                     /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(429..448)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1039..1058
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1357..1376)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1401..1448
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1777..2365
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2266..2285
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2540..2843
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2844..3047
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2998..3018
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(4718..4720,4721..5434)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4718..4720,4721..5431)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4760..4781)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5021..5040)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5368..5389
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5557..5678
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5594..5613)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5648..5667
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5685..6140)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5822..5843)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6034..6053
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6167..6271
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6273..6290,1..340)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        join(6273..6290,1..174)
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
ORIGIN
        1 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
       61 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
      121 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
      181 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
      241 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
      301 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
      361 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
      421 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
      481 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
      541 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
      601 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
      661 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
      721 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
      781 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
      841 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
      901 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
      961 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     1021 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     1081 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     1141 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     1201 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     1261 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     1321 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     1381 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     1441 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     1501 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     1561 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     1621 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     1681 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     1741 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     1801 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     1861 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     1921 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     1981 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2041 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2101 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     2161 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     2221 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     2281 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     2341 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     2401 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     2461 ccgtattacc gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata
     2521 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     2581 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     2641 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     2701 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     2761 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     2821 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     2881 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     2941 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     3001 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc
     3061 gtcagatccg ctagcgctac cggactcaga tctcgagctc aagcttcgaa ttcccaccat
     3121 ggccttcttg gacaatccaa ctatcattct agctcatatt cgacagtcac atgtgaccag
     3181 tgatgacacg ggaatgtgtg agatggttct cattgatcat gatgttgacc tagagaagat
     3241 tcatcctcct tcaatgcctg gagacagtgg gtcagaaatt cagggaagca atggtgagac
     3301 tcagggctat gtatatgccc agtcagtcga tattacctca agttgggact ttggtattag
     3361 aagacgctca aacacagctc aaagattaga acgactccga aaagagagac aaaaccagat
     3421 caaatgcaaa aatattcagt ggaaagaaag aaattctaag caatcagccc aggagttaaa
     3481 gtcactgttt gaaaaaaaat ctctcaaaga gaagcctcca atttctggga agcagtcgat
     3541 attatctgta cgcctagaac agtgccctct gcagctgaat aaccctttta acgagtattc
     3601 caaatttgat ggcaagggtc atgtaggtac aacagcaacc aagaagatcg atgtctacct
     3661 ccctctgcac tcgagccagg acagactgct gccaatgacc gtggtgacaa tggccagcgc
     3721 cagggtgcag gacctgatcg ggctcatctg ctggcagtat acaagcgaag gacgggagcc
     3781 gaagctcaat gacaatgtca gtgcctactg cctgcatatt gctgaggatg atggggaggt
     3841 ggacaccgat ttccccccgc tggattccaa tgagcccatt cataagtttg gcttcagtac
     3901 tttggccctg gttgaaaagt actcatctcc tggtctgaca tccaaagagt cactctttgt
     3961 tcgaataaat gctgctcatg gattctccct tattcaggtg gacaacacaa aggttaccat
     4021 gaaggaaatc ttactgaagg cagtgaagcg aagaaaagga tcccagaaag tttcaggccc
     4081 tcagtaccgc ctggagaagc agagcgagcc caatgtcgcc gttgacctgg acagcacttt
     4141 ggagagccag agcgcatggg agttctgcct ggtccgcgag aacagttcaa gggcagacgg
     4201 ggtttttgag gaggattcgc aaattgacat agccacagta caggatatgc ttagcagcca
     4261 ccattacaag tcattcaaag tcagcatgat ccacagactg cgattcacaa ccgacgtaca
     4321 gctaggtatc tctggagaca aagtagagat agaccctgtt acgaatcaga aagccagcac
     4381 taagttttgg attaagcaga aacccatctc aatcgattcc gacctgctct gtgcctgtga
     4441 ccttgctgaa gagaaaagcc ccagtcacgc aatatttaaa ctcacgtatc taagcaatca
     4501 cgactataaa cacctctact ttgaatcgga cgctgctacc gtcaatgaaa ttgtgctcaa
     4561 ggttaactac atcctggaat cgcgagctag cactgcccgg gctgactact ttgctcaaaa
     4621 acaaagaaaa ctgaacagac gtacgagctt cagcttccag aaggagaaga aatccgggca
     4681 gcagccgcgg gcccgggatc caccggtcgc aaccttggtg agcaagggcg aggagctgtt
     4741 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     4801 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     4861 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     4921 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     4981 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     5041 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     5101 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     5161 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     5221 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     5281 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     5341 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     5401 gatcactctc ggcatggacg agctgtacaa gtaaagcggc cgcgactcta gatcataatc
     5461 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     5521 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     5581 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5641 tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa
     5701 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     5761 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     5821 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     5881 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     5941 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     6001 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     6061 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     6121 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     6181 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     6241 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca
//
