LOCUS       sequence-174183-        6182 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP-tagged mSin1 (isoform 2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6182)
  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 6182)
  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-mSin1.2-GFP
FEATURES             Location/Qualifiers
     source          1..6182
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        37..394
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        37..228
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(60..80)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      245..380
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             429..1223
                     /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             429..1223
                     /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(483..502)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1093..1112
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1411..1430)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1455..1502
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1831..2419
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2320..2339
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2594..2897
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2898..3101
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3052..3072
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(4664..4666,4667..5380)
                     /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(4664..4666,4667..5377)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4706..4727)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4967..4986)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5314..5335
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5503..5624
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5540..5559)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5594..5613
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5631..6086)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5768..5789)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5980..5999
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(6113..6182,1..35)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg
       61 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
      121 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
      181 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
      241 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
      301 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
      361 aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat
      421 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
      481 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
      541 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
      601 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
      661 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
      721 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
      781 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
      841 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
      901 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca
      961 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     1021 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     1081 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     1141 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     1201 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     1261 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     1321 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     1381 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg
     1441 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt
     1501 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca
     1561 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga
     1621 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc
     1681 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     1741 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     1801 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     1861 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     1921 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     1981 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     2041 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     2101 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     2161 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     2221 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     2281 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     2341 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     2401 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     2461 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     2521 taccgccatg cattagttat taatagtaat caattacggg gtcattagtt catagcccat
     2581 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     2641 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     2701 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     2761 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     2821 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     2881 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     2941 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     3001 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     3061 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga
     3121 tccgctagcg ctaccggact cagatctcga gctcaagctt cgaattccca ccatggcctt
     3181 cttggacaat ccaactatca ttctagctca tattcgacag tcacatgtga ccagtgatga
     3241 cacgggaatg tgtgagatgg ttctcattga tcatgatgtt gacctagaga agattcatcc
     3301 tccttcaatg cctggagaca gtgggtcaga aattcaggga agcaatggtg agactcaggg
     3361 ctatgtatat gcccagtcag tcgatattac ctcaagttgg gactttggta ttagaagacg
     3421 ctcaaacaca gctcaaagat tagaacgact ccgaaaagag agacaaaacc agatcaaatg
     3481 caaaaatatt cagtggaaag aaagaaattc taagcaatca gcccaggagt taaagtcact
     3541 gtttgaaaaa aaatctctca aagagaagcc tccaatttct gggaagcagt cgatattatc
     3601 tgtacgccta gaacagtgcc ctctgcagct gaataaccct tttaacgagt attccaaatt
     3661 tgatggcaag ggtcatgtag gtacaacagc aaccaagaag atcgatgtct acctccctct
     3721 gcactcgagc caggacagac tgctgccaat gaccgtggtg acaatggcca gcgccagggt
     3781 gcaggacctg atcgggctca tctgctggca gtatacaagc gaaggacggg agccgaagct
     3841 caatgacaat gtcagtgcct actgcctgca tattgctgag gatgatgggg aggtggacac
     3901 cgatttcccc ccgctggatt ccaatgagcc cattcataag tttggcttca gtactttggc
     3961 cctggttgaa aagtactcat ctcctggtct gacatccaaa gagtcactct ttgttcgaat
     4021 aaatgctgct catggattct cccttattca ggtggacaac acaaaggtta ccatgaagga
     4081 aatcttactg aaggcagtga agcgaagaaa aggatcccag aaagtttcag gttcaagggc
     4141 agacggggtt tttgaggagg attcgcaaat tgacatagcc acagtacagg atatgcttag
     4201 cagccaccat tacaagtcat tcaaagtcag catgatccac agactgcgat tcacaaccga
     4261 cgtacagcta ggtatctctg gagacaaagt agagatagac cctgttacga atcagaaagc
     4321 cagcactaag ttttggatta agcagaaacc catctcaatc gattccgacc tgctctgtgc
     4381 ctgtgacctt gctgaagaga aaagccccag tcacgcaata tttaaactca cgtatctaag
     4441 caatcacgac tataaacacc tctactttga atcggacgct gctaccgtca atgaaattgt
     4501 gctcaaggtt aactacatcc tggaatcgcg agctagcact gcccgggctg actactttgc
     4561 tcaaaaacaa agaaaactga acagacgtac gagcttcagc ttccagaagg agaagaaatc
     4621 cgggcagcag ccgcgggccc gggatccacc ggtcgcaacc ttggtgagca agggcgagga
     4681 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     4741 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     4801 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     4861 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     4921 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     4981 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     5041 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     5101 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     5161 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     5221 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     5281 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     5341 cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc
     5401 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     5461 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     5521 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5581 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag
     5641 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     5701 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     5761 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     5821 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     5881 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     5941 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     6001 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     6061 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     6121 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     6181 ct
//
