LOCUS       sequence-178993-        5427 bp    DNA     circular SYN 14-APR-2025
DEFINITION  To express GFP-tagged CAHS1 in mammalian cells transiently.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5427)
  AUTHORS   Yamaguchi A, Tanaka S, Yamaguchi S, Kuwahara H, Takamura C, 
            Imajoh-Ohmi S, Horikawa DD, Toyoda A, Katayama T, Arakawa K, 
            Fujiyama A, Kubo T, Kunieda T
  TITLE     Two novel heat-soluble protein families abundantly expressed in an 
            anhydrobiotic tardigrade.
  JOURNAL   PLoS One. 2012;7(8):e44209.
  PUBMED    22937162
REFERENCE   2  (bases 1 to 5427)
  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: "PLoS One.";
            date: "2012"; volume: "7(8)"; pages: "e44209"
COMMENT     Sequence Label: pAcGFP1-N1-CAHS1
FEATURES             Location/Qualifiers
     source          1..5427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     78..97
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        211..315
                     /gene="bla"
                     /label=AmpR promoter
     promoter        317..674
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        317..508
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(340..360)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      525..660
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             709..1503
                     /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             709..1503
                     /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(763..782)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1373..1392
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1691..1710)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1735..1782
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2111..2699
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2600..2619
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2874..3177
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3178..3381
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3332..3352
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4187..4906
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     polyA_signal    5028..5149
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5065..5084)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5119..5138
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(join(5156..5427,1..184))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5293..5314)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
       61 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
      121 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
      181 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
      241 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
      301 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
      361 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
      421 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
      481 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
      541 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
      601 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
      661 taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
      721 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
      781 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
      841 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca
      901 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
      961 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     1021 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     1081 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     1141 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     1201 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc
     1261 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     1321 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     1381 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     1441 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     1501 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     1561 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     1621 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg
     1681 ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa
     1741 taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt
     1801 cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg
     1861 tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca
     1921 acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt tagattgatt
     1981 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2041 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2101 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2161 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2221 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     2281 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2341 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2401 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2461 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2521 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2581 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2641 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2701 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2761 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat
     2821 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     2881 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     2941 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     3001 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     3061 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     3121 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     3181 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     3241 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     3301 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     3361 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact
     3421 cagatctcga gctcaagctt cgaattctgc agtcgacggt accatgcctt acgaaaagca
     3481 cgttgaacag acggtggtgg aaaaaactga gcagcctgga cactcgtcga cgcaccatgc
     3541 tcccgcccaa aggaccgtag ctcgcgagca ggaggaagtt gtccacaaag agtttaccca
     3601 taccgacatt cgagttcccc acatcgacgc acctcctcca atcatcgctg ccagcgcagt
     3661 gggcttggcc gaggagatcg tcagtcatgg tttccaagcc tcggcggccc gcatcagcgg
     3721 tgcttccacc gaggtcgata tgcgcccaag tcccaagcta gccgaggaag ctcgtcgtga
     3781 tgccgaacga tatcaaaagg agcacgagat gatcaacaga caagccgaag ccacgctgca
     3841 aaagaaggcg gaggagtacc gtcaccagac tgaggcggaa gccgagaaga ttcgccgcga
     3901 actggaaaag cagcatgaac gggacatcca gttcaggaag gacctcatcg atcagaccat
     3961 cgaaaagcag aagcgcgaag ttgatctgga agccaagatg gccaaacgcg agctggatcg
     4021 cgaagcgcag ttggctaagg aagccctgga acgttctcga atggccacca acgtggaagt
     4081 cacgctggat accgcagcgg gacatacagt aagtggggga actaccgtct ccagcgtcga
     4141 caaagttgaa accgtccgcg agcgcaaaca tcatgatcca ccggtcatgg tgagcaaggg
     4201 cgccgagctg ttcaccggca tcgtgcccat cctgatcgag ctgaatggcg atgtgaatgg
     4261 ccacaagttc agcgtgagcg gcgagggcga gggcgatgcc acctacggca agctgaccct
     4321 gaagttcatc tgcaccaccg gcaagctgcc tgtgccctgg cccaccctgg tgaccaccct
     4381 gagctacggc gtgcagtgct tctcacgcta ccccgatcac atgaagcagc acgacttctt
     4441 caagagcgcc atgcctgagg gctacatcca ggagcgcacc atcttcttcg aggatgacgg
     4501 caactacaag tcgcgcgccg aggtgaagtt cgagggcgat accctggtga atcgcatcga
     4561 gctgaccggc accgatttca aggaggatgg caacatcctg ggcaataaga tggagtacaa
     4621 ctacaacgcc cacaatgtgt acatcatgac cgacaaggcc aagaatggca tcaaggtgaa
     4681 cttcaagatc cgccacaaca tcgaggatgg cagcgtgcag ctggccgacc actaccagca
     4741 gaataccccc atcggcgatg gccctgtgct gctgcccgat aaccactacc tgtccaccca
     4801 gagcgccctg tccaaggacc ccaacgagaa gcgcgatcac atgatctact tcggcttcgt
     4861 gaccgccgcc gccatcaccc acggcatgga tgagctgtac aagtgagcgg ccgcgactct
     4921 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     4981 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     5041 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5101 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt
     5161 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     5221 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     5281 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     5341 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     5401 agttttttgg ggtcgaggtg ccgtaaa
//
