LOCUS       sequence-356540-        7534 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses SMCR8-tdTomato in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7534)
  AUTHORS   Amick J, Roczniak-Ferguson A, Ferguson SM
  TITLE     C9orf72 binds SMCR8, localizes to lysosomes and regulates mTORC1 
            signaling.
  JOURNAL   Mol Biol Cell. 2016 Aug 24. pii: mbc.E16-01-0003.
  PUBMED    27559131
REFERENCE   2  (bases 1 to 7534)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Biol 
            Cell. 2016 Aug 24. pii: mbc.E16-01-0003."
COMMENT     Sequence Label: ptdTomato-SMCR8
FEATURES             Location/Qualifiers
     source          1..7534
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        68..371
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        372..575
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     526..546
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3497..3506
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3503..4204
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(3636..3654)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3904..3923
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4098..4121
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4329..4450
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4366..4385)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4420..4439
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4457..4912)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4594..4615)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4806..4825
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4939..5043
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5045..5402
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5045..5236
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5068..5088)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5253..5388
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5437..6231
                     /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             5437..6231
                     /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(5491..5510)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6101..6120
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6419..6438)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6463..6510
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6839..7427
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7328..7347
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
       61 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      121 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      181 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      241 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      301 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      361 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      421 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      481 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      541 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      601 agcgctaccg gactcagatc tcgagctcaa gcttgccacc atgatcagcg cccctgacgt
      661 agtggccttc accaaagagg aagagtatga agaagagcct tacaatgagc cggccctgcc
      721 tgaggagtac tcggtgccgc tcttcccctt cgccagtcag ggtgctaacc cctggtcaaa
      781 actgtccggg gccaagtttt cgagggactt cattcttatt tccgagttct ctgagcaggt
      841 gggaccccaa cccttactga ccatccccaa tgacaccaaa gtttttggca cttttgatct
      901 caattacttc tccctgcgta tcatgtctgt ggattaccag gcttccttcg tgggccatcc
      961 tcctggatct gcctacccca agctgaactt cgtggaggac tccaaggtgg tgctgggaga
     1021 ttctaaggag ggcgcctttg catacgtgca ccaccttacc ctatacgacc tggaggcccg
     1081 tggcttcgtg aggccgtttt gcatggctta tatctctgca gaccagcata aaatcatgca
     1141 gcagttccag gagctttcag ccgaattttc cagagcttct gagtgcttga agactggcaa
     1201 caggaaggca tttgctgggg aacttgaaaa aaagctgaaa gacttggatt acaccaggac
     1261 agtgctacac acagaaacgg agatccagaa gaaagccaac gacaaaggct tttactcatc
     1321 tcaggcaatt gagaaagcca atgaactggc cagtgtggag aagtccatca ttgaacatca
     1381 agacctgctg aagcagatcc gctcataccc tcatcggaag ttgaaggggc atgatttgtg
     1441 tcctggtgag atggagcaca tccaggatca ggccagccag gcatccacta cctctaaccc
     1501 tgatgagtct gccgacacag acctttacac ctgcagacca gcctacaccc caaaacttat
     1561 caaagcaaag tccaccaagt gttttgacaa gaagttgaag accttggagg agctctgtga
     1621 cactgaatat ttcacccaga ccctggctca gctcagccac attgaacaca tgttcagagg
     1681 agacctgtgt tacctcctga ccagtcagat tgatagagca cttctaaaac aacagcatat
     1741 aacaaacttt ctctttgaag actttgtgga ggtcgatgac aggatggtgg agaaacaaga
     1801 aagcataccc tctaagccca gtcaagacag gccgccttcc agttctctag aagaatgccc
     1861 aattcctaaa gtgttaatta gtgttggttc ttacaagtcc agtgtggagt ctgtgttgat
     1921 caagatggag caggaactgg gagatgagga gtacaaggaa gtggaagtga ctgagttgag
     1981 cagtttcgac ccccaggaaa acttggacta cctggatatg gatatgaaag ggagtatcag
     2041 cagtggtgaa agtattgaag ttttgggcac ggagaaatcc acctccgtgc tttctaaatc
     2101 tgacagccag gcaagcctca cagtaccatt gagcccccag gtggtccgga gcaaagcagt
     2161 cagccacagg accatcagcg aggacagtat tgaagtcctc agtacctgcc cctctgaggc
     2221 cctcatccct gatgacttta aggccagcta cccaagtgcc attaatgaag aagaatcata
     2281 tccagatggc aatgaaggag ccatccgctt ccaggcaagc atcagtcctc cagaactggg
     2341 tgagacagag gaaggcagca tagaaaacac cccatcacaa atagactcct cctgctgtat
     2401 tgggaaggag agcgatggtc agttggtgct gccctccact ccagcccaca cacactctga
     2461 cgaggatggg gtggtgagca gccccccaca gcgccacagg cagaaggacc aggggttccg
     2521 tgtagacttt tcagtggaaa atgccaaccc ttcttcccga gacaacagtt gtgaagggtt
     2581 tcccgcttat gagctggacc cgagccacct gctggctagc cgggacatca gtaagaccag
     2641 cctggacaac tactcagaca ccaccagcta cgtgagcagt gtagcgtcca ccagctcaga
     2701 caggatcccc tctgcttatc ctgctggcct gtcttccgat aggcataaaa agagggctgg
     2761 ccagaacgcc ttaaaattca tccgccagta cccctttgcc cacccagcca tctactccct
     2821 gctcagtggg aggacacttg tggtcctggg ggaagatgag gccatagtca ggaaactcgt
     2881 gactgcactg gctatctttg tccccagcta tggctgctac gctaagcccg tgaaacattg
     2941 ggcctcctcc cctttgcaca ttatggattt tcagaagtgg aagcttattg gcttgcagag
     3001 agtggcctcc cctgccggtg ccggtaccct ccatgccctg agccgctaca gccgctacac
     3061 gagcatcctg gaccttgaca acaaaaccct gcgctgcccc ctttacagag gcaccctggt
     3121 gccccgcctg gcagaccacc gcacacagat caagcggggc agcacctact acctgcatgt
     3181 ccagagcatg ctcacccagc tctgctccaa ggccttcctc tacaccttct gccaccacct
     3241 gcacctgcct acccacgaca aggagacaga ggagctggta gccagccgcc agatgagctt
     3301 cctaaagctg accctgggtc tggtgaatga ggatgttagg gtggtccagt acctggctga
     3361 gctgctgaag ctgcactaca tgcaggaatc tccagggacc agccacccca tgctcaggtt
     3421 tgactatgtc cccagctttt tgtataaaat ccgaattctg cagtcgacgg taccgcgggc
     3481 ccgggatcca ccggtcgcca ccatggtgag caagggcgag gaggtcatca aagagttcat
     3541 gcgcttcaag gtgcgcatgg agggctccat gaacggccac gagttcgaga tcgagggcga
     3601 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg
     3661 ccccctgccc ttcgcctggg acatcctgtc cccccagttc atgtacggct ccaaggcgta
     3721 cgtgaagcac cccgccgaca tccccgatta caagaagctg tccttccccg agggcttcaa
     3781 gtgggagcgc gtgatgaact tcgaggacgg cggtctggtg accgtgaccc aggactcctc
     3841 cctgcaggac ggcacgctga tctacaaggt gaagatgcgc ggcaccaact tcccccccga
     3901 cggccccgta atgcagaaga agaccatggg ctgggaggcc tccaccgagc gcctgtaccc
     3961 ccgcgacggc gtgctgaagg gcgagatcca ccaggccctg aagctgaagg acggcggcca
     4021 ctacctggtg gagttcaagt ctatctacat ggccaagaag cccgtgcaac tgcccggcta
     4081 ctactacgtg gacaccaagc tggacatcac ctcccacaac gaggactaca ccatcgtgga
     4141 acagtacgag cgctccgagg gccgccacca cctgttcctg tacggcatgg acgagctgta
     4201 caagtaggcg gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac
     4261 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     4321 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4381 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4441 atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     4501 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     4561 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     4621 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     4681 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     4741 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     4801 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     4861 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
     4921 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4981 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5041 agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     5101 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     5161 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     5221 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     5281 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     5341 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     5401 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     5461 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     5521 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     5581 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     5641 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     5701 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     5761 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     5821 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     5881 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     5941 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     6001 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     6061 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     6121 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     6181 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     6241 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     6301 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     6361 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     6421 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     6481 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     6541 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     6601 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     6661 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     6721 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6781 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6841 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6901 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6961 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     7021 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     7081 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     7141 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     7201 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     7261 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     7321 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     7381 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     7441 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     7501 tatcccctga ttctgtggat aaccgtatta ccgc
//
