LOCUS       sequence-178999-        5595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express GFP-tagged RvLEAM in mammalian cells transiently.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5595)
  AUTHORS   Tanaka S, Tanaka J, Miwa Y, Horikawa DD, Katayama T, Arakawa K, 
            Toyoda A, Kubo T, Kunieda T
  TITLE     Novel mitochondria-targeted heat-soluble proteins identified in the 
            anhydrobiotic Tardigrade improve osmotic tolerance of human cells.
  JOURNAL   PLoS One. 2015 Feb 12;10(2):e0118272. eCollection 2015.
  PUBMED    25675104
REFERENCE   2  (bases 1 to 5595)
  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: "PLoS One.";
            date: "2015-02-12"; volume: "10(2)"; pages: "e0118272. eCollection 
            2015"
COMMENT     Sequence Label: pAcGFP1-N1-RvLEAM
FEATURES             Location/Qualifiers
     source          1..5595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        26..130
                     /gene="bla"
                     /label=AmpR promoter
     promoter        132..489
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        132..323
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(155..175)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      340..475
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             524..1318
                     /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             524..1318
                     /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(578..597)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1188..1207
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1506..1525)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1550..1597
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1926..2514
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2415..2434
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2689..2992
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2993..3196
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3147..3167
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4170..4889
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     polyA_signal    5011..5132
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5048..5067)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5102..5121
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5139..5594)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5276..5297)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5488..5507
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
       61 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
      121 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
      181 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
      241 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
      301 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
      361 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
      421 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
      481 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
      541 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
      601 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
      661 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
      721 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
      781 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
      841 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
      901 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
      961 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     1021 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     1081 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     1141 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     1201 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     1261 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     1321 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     1381 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     1441 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     1501 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     1561 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     1621 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     1681 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     1741 ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa
     1801 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1861 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1921 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1981 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2041 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     2101 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     2161 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2221 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2281 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2341 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2401 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2461 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2521 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2581 cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta gttattaata
     2641 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     2701 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     2761 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     2821 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     2881 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     2941 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     3001 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     3061 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     3121 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     3181 ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcgctacc ggactcagat
     3241 ctcgagctca agcttcgaat tctgcagtcg acggtaccat gtttctcgcc cgaaacgctg
     3301 gacgcgctgg atatcgaggt gttgtagctt accagcaagc agcctctttc tctgtttcat
     3361 cagctaaggc tgctggatca cggtcaagcg gtggctccga cgctggcgat tatgctaggg
     3421 aggccgcgga acatgccaaa gctggattga aggatctcaa gaatgaagcc tcgtggaaag
     3481 ccaaaggtgt cgctaaccaa gccgctggag cattcgaacg agcgaaggat acagtgaagg
     3541 aaggtgtcca tgacatgaag cgcagtggaa gccgcgtctt cgaacaagga caggaggaag
     3601 tagaggctgg tgctcaacac gcaaaggctg gctatcagag tgccaagaat gctgcgcaag
     3661 acactgccgc aactcttaaa gacaaagccg gcagcgcctg gaatcaagcg aaacacgttg
     3721 tggaagataa gggagaggat gttgttgaag cagtcaagga cactgcttca aaagtctggg
     3781 gcaaagccaa acatgtggcc gaggacgtta aggagaacgc gcaatcacca ggtggcattg
     3841 cggataaggc ctccgatgtg tggtcagcgg ccaaggataa agctgctgat gtcttgtcgg
     3901 gagctaagca cactgctgag aacctagctc acaaagccca agccgccatc cacgatgcta
     3961 cagcttcttc aggctcgcag tcgcagtcgc agtcccagtc tcagtatcga caaggacaac
     4021 aacagggaag acaagatcag cagcaaagca agagccagtg gggacagact agcccgcagt
     4081 ctccggatgg attccgccct caagccggcc agggaccaca aggaggcaag ggacctggtc
     4141 aagctggcgg tcgacgagat ccaccggtca tggtgagcaa gggcgccgag ctgttcaccg
     4201 gcatcgtgcc catcctgatc gagctgaatg gcgatgtgaa tggccacaag ttcagcgtga
     4261 gcggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     4321 ccggcaagct gcctgtgccc tggcccaccc tggtgaccac cctgagctac ggcgtgcagt
     4381 gcttctcacg ctaccccgat cacatgaagc agcacgactt cttcaagagc gccatgcctg
     4441 agggctacat ccaggagcgc accatcttct tcgaggatga cggcaactac aagtcgcgcg
     4501 ccgaggtgaa gttcgagggc gataccctgg tgaatcgcat cgagctgacc ggcaccgatt
     4561 tcaaggagga tggcaacatc ctgggcaata agatggagta caactacaac gcccacaatg
     4621 tgtacatcat gaccgacaag gccaagaatg gcatcaaggt gaacttcaag atccgccaca
     4681 acatcgagga tggcagcgtg cagctggccg accactacca gcagaatacc cccatcggcg
     4741 atggccctgt gctgctgccc gataaccact acctgtccac ccagagcgcc ctgtccaagg
     4801 accccaacga gaagcgcgat cacatgatct acttcggctt cgtgaccgcc gccgccatca
     4861 cccacggcat ggatgagctg tacaagtgag cggccgcgac tctagatcat aatcagccat
     4921 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     4981 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     5041 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5101 tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt
     5161 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     5221 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     5281 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     5341 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     5401 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     5461 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     5521 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     5581 gctacagggc gcgtc
//
