LOCUS       sequence-417775-        6183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mScarlet-I-CCM2 targeted to the mitochondria in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6183)
  AUTHORS   Ball NJ, Ghimire S, Follain G, Pajari AO, Wurzinger D, Vaitkeviciute
            M, Cowell AR, Berki B, Ivaska J, Paatero I, Goult BT, Jacquemet G
  TITLE     TLNRD1 is a CCM complex component and regulates endothelial barrier 
            integrity.
  JOURNAL   J Cell Biol. 2024 Sep 2;223(9):e202310030. doi: 
            10.1083/jcb.202310030. Epub 2024 Jul 16.
  PUBMED    39013281
REFERENCE   2  (bases 1 to 6183)
  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."; date: "2024-09-2"; volume: "223(9):e202310030. doi"; pages: 
            " 10.1083/jcb.202310030. Epub 2024 Jul 16"
COMMENT     Sequence Label: mito-CCM2-mScarlet-I
FEATURES             Location/Qualifiers
     source          1..6183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(27..46)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     637..656
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(955..974)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    999..1046
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1375..1963
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1864..1883
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2138..2441
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2442..2645
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2596..2616
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2690..2709
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2690..2708
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2728..2802
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      2830..2839
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2836..3531
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2969..2987)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3237..3256
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    5048..5169
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5085..5104)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5139..5158
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5176..5631)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5313..5334)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5525..5544
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5658..5762
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5764..6121
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5764..5955
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5787..5807)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5972..6107
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(6156..6183,1..767)
                     /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             join(6156..6183,1..767)
                     /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"
ORIGIN
        1 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
       61 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
      121 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
      181 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
      241 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
      301 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
      361 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
      421 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
      481 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
      541 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
      601 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
      661 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
      721 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
      781 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
      841 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
      901 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac
      961 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     1021 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     1081 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     1141 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     1201 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     1261 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     1321 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     1381 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     1441 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     1501 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     1561 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     1621 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     1681 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     1741 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     1801 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     1861 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     1921 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     1981 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     2041 ccctgattct gtggataacc gtattaccgc catgcattag ttattaatag taatcaatta
     2101 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     2161 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     2221 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     2281 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     2341 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     2401 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     2461 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     2521 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     2581 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     2641 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     2701 actataggga gacccaagct ggctagcatg tccgtcctga cgccgctgct gctgcggggc
     2761 ttgacaggct cggcccggcg gctcccagtg ccgcgcgcca agatccattc gttgggggat
     2821 ccaccggtcg ccaccatggt gagcaagggc gaggcagtga tcaaggagtt catgcggttc
     2881 aaggtgcaca tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag
     2941 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg
     3001 cccttctcct gggacatcct gtcccctcag ttcatgtacg gctccagggc cttcatcaag
     3061 caccccgccg acatccccga ctactataag cagtccttcc ccgagggctt caagtgggag
     3121 cgcgtgatga acttcgagga cggcggcgcc gtgaccgtga cccaggacac ctccctggag
     3181 gacggcaccc tgatctacaa ggtgaagctc cgcggcacca acttccctcc tgacggcccc
     3241 gtaatgcaga agaagacaat gggctgggaa gcgtccaccg agcggttgta ccccgaggac
     3301 ggcgtgctga agggcgacat taagatggcc ctgcgcctga aggacggcgg ccgctacctg
     3361 gcggacttca agaccaccta caaggccaag aagcccgtgc agatgcccgg cgcctacaac
     3421 gtcgaccgca agttggacat cacctcccac aacgaggact acaccgtggt ggaacagtac
     3481 gaacgctccg agggccgcca ctccaccggc ggcatggacg agctgtacaa gtccggactc
     3541 agatctcgag ctatggagga agaaggcaaa aagggcaaaa aaccgggaat agtatcacct
     3601 ttcaagcggg tctttctgaa gggggagaaa tcacgggata aaaaagccca tgagaaagtt
     3661 accgaacggc ggcctctcca tactgttgtc ttgtctctcc cagaaagagt cgagcctgac
     3721 agactgcttt ccgattatat tgaaaaagaa gtaaaatacc tgggacagtt gaccagcatc
     3781 cctgggtatc ttaaccccag cagtaggacg gaaattcttc attttataga caacgcaaaa
     3841 agagctcacc aattgccggg acacctcacc caggagcacg acgcggtcct gagtctgtca
     3901 gcttacaacg taaagctcgc gtggcgggac ggagaagaca tcatcctgcg ggtacctatt
     3961 catgatattg ccgctgtctc ctatgtaagg gatgacgccg cgcacttggt agtccttaag
     4021 accgcacaag acccgggtat aagtccgtca caatccctct gtgcggaaag ttcccggggg
     4081 ctgagtgcgg gctcactctc tgagagcgca gtgggccccg tcgaggcctg ttgcctcgtt
     4141 attctcgcgg ccgagtccaa agttgccgca gaagaacttt gctgtctgtt ggggcaggtt
     4201 ttccaagtag tctatactga gagcacaatt gatttccttg atagagcaat ctttgatgga
     4261 gctagtaccc ctacgcacca cttgtctctg cattccgacg atagcagtac taaagttgat
     4321 atcaaagaga cttatgaggt tgaggcttcc accttttgtt ttcctgaatc cgtcgatgtg
     4381 ggaggggcaa gcccccacag caagactatc agcgaatccg aactctcagc atctgcaaca
     4441 gaactgctcc aggactatat gctcacgttg cggacaaaac tctcttctca ggagattcag
     4501 cagtttgctg ccctgttgca tgaatatagg aatggtgcga gcattcacga gttctgcatt
     4561 aatttgaggc agttgtatgg ggattctcgc aaatttcttc tgctcggcct tagacccttt
     4621 atcccagaaa aagattcaca gcatttcgag aactttctgg agactattgg cgtcaaagac
     4681 ggtcgcggaa taattacaga ctcatttggc aggcacaggc gggctctcag tacgaccagt
     4741 tcctcaacga ccaacggcaa ccgcgcaacg ggttcttctg atgataggtc cgccccctct
     4801 gaaggtgatg aatgggacag aatgataagc gatattagct cagacatcga agccctcgga
     4861 tgttccatgg accaggattc tgcataggaa ttctgcagtc gacggtaccg cgggcccggg
     4921 atccaccgga tctagataac tgatcataat cagccatacc acatttgtag aggttttact
     4981 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     5041 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     5101 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     5161 tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     5221 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     5281 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     5341 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     5401 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     5461 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     5521 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     5581 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca
     5641 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5701 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5761 gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     5821 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     5881 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     5941 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     6001 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     6061 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     6121 agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     6181 cag
//
