LOCUS       sequence-391227-        6686 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3xHA-3'UTRmg105 -pmg153-NAT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6686)
  AUTHORS   Philippe N, Shukla A, Abergel C, Bisio H
  TITLE     Genetic manipulation of giant viruses and their host, Acanthamoeba 
            castellanii.
  JOURNAL   Nat Protoc. 2023 Nov 14. doi: 10.1038/s41596-023-00910-y.
  PUBMED    37964008
REFERENCE   2  (bases 1 to 6686)
  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: "Nat Protoc.
            2023 Nov 14. doi: 10.1038/s41596-023-00910-y."
COMMENT     Sequence Label: vAS1
FEATURES             Location/Qualifiers
     source          1..6686
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             744..1421
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             744..1421
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(874..892)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1142..1161
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1336..1359
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     rep_origin      1621..2076
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1708..1727)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1918..1939
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2193..2553
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2193..2384
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2216..2236)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             2740..2829
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3494..4066
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     primer_bind     complement(4630..4649)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4749..4771
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4809..4827)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4895..4999
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5000..5068,5069..5860)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5218..5237)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6031..6619
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6520..6539
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatctatat gcaaccctgt gacgagagac agggagtgct gttagagaag agcgcacatt
       61 cagcgctcaa gtggtcttgc gtttggtgtc agcacgaggt gcgccagacc caggtcgcac
      121 gcaggcccct cagcgcggtc gccgtgtgtt ggtattgtgt gcctgagtga tgcggaatgc
      181 gttctgcttg taggcgtacg agaccgacgc caggcggcct gtcgtgctgg gctcgtccgc
      241 ggcaggctct gtagggctgg cgcggagtgg gcgatcgtcc tcccgccgcg aggaaacagc
      301 aacggacagg cttcattgcc acctcatcgc cgctttcgcg ctcgaggcca gggcgtggcg
      361 agcaggtaag aatctgagaa gttgaattgt gaattaacac aaagaagacg aacgaacccg
      421 ccgtggtatg gaagaaggcc acctcaacca atctcaagat cacctcctaa tcgtccaacc
      481 cggcctacaa tcgcgcacat cagcactcga tctcgcactt cggtccgttt ggccctcgag
      541 ccaaagccaa gcagttttcc aggcaccgtt gaagtgcgaa tagatgaaat ttccggtcta
      601 ccccaatgac atggcgtttt gtcacaacac atggcgctgt gctggtggtc tcggtggggc
      661 ctatttattt ggagttgttt gcttgctctc tttcatcttt gtcttcctac acagcagcaa
      721 acaccactct ctcactaaga catatggcct cctccgagga cgtcatcaag gagttcatgc
      781 gcttcaaggt gcgcatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
      841 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc
      901 ccctgccctt cgcctgggac atcctgtccc ctcagttcca gtacggctcc aaggcctacg
      961 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     1021 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     1081 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     1141 gccccgtaat gcagaagaag accatgggct gggaggcctc caccgagcgg atgtaccccg
     1201 aggacggcgc cctgaagggc gagatcaaga tgaggctgaa gctgaaggac ggcggccact
     1261 acgacgccga ggtcaagacc acctacatgg ccaagaagcc cgtgcagctg cccggcgcct
     1321 acaagaccga catcaagctg gacatcacct cccacaacga ggactacacc atcgtggaac
     1381 agtacgagcg cgccgagggc cgccactcca ccggcgccta atctagagtc gacccaacaa
     1441 caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag
     1501 caagtaaaac ctctacaaat gtggtaaaat ccgataagga tcgatccggg ctggcgtaat
     1561 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     1621 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1681 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1741 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1801 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1861 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1921 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1981 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2041 acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac
     2101 gcatctgtgc ggtatttcac accgcatacg cggatctgcg cagcaccatg gcctgaaata
     2161 acctctgaaa gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat
     2221 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2281 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2341 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     2401 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     2461 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     2521 ggcttttttg gaggatcaaa cgacgccttg caacaagctg agaaaaaacc aggatcggca
     2581 aggaaggatt ttcaacggaa acgttggcat caccgggtat aaaaaggggc caattttttt
     2641 gttgatttgt tgcgcgaatt cttgctttcg gcatcgaatt caagggagaa ggagtcgatt
     2701 cacacataca acaagatcct aggcttttgc aaaaagcttt acccatacga tgttcctgac
     2761 tatgcgggct atccgtatga cgtcccggac tatgcaggtt cctatccata tgacgttcca
     2821 gattacgctt gataattttt taataaatat taaaaaatta tcaatcaatg aatttgacag
     2881 catctgtaat tttatgtatc aaactatcat aatcataaat accaaaataa tcaaaatatt
     2941 ctaaattttt ccaaaatatt agcaatttta tcatatattt tagttttatt aataattagt
     3001 gtgtgacaac ttttacatac tcttttttta ttttttttta gtgaagtgat atagtaagac
     3061 atgttccaat aatcggccgg atcaggacga tctaagaaat cgggaattac aatgctgtat
     3121 cctgcgcatt tataacaaaa tatattgcca caacttcggc aatgatgttt gcgtaaaaat
     3181 gcaccaaatg ttgcattaca accatgacac gaggatactc tatgatcctc gacccaaata
     3241 ttatttttat taccaataac aatagatgtt gttgtttttt cactcattat attattaata
     3301 taataatata cttatattgt ttgaatagat ctaacattat ctgttaaaaa aattcgaaaa
     3361 aaaattgaaa aaaatatgta ttgatatagt taatgatata aaaacgtcat tatatcatta
     3421 atacatcgaa tatcgactta cttgatcctt ctgacacaac agtctcgaac ttaaggctag
     3481 agccaccgat atcatgggta ccactcttga cgacacggct taccggtacc gcaccagtgt
     3541 cccgggggac gccgaggcca tcgaggcact ggatgggtcc ttcaccaccg acaccgtctt
     3601 ccgcgtcacc gccaccgggg acggcttcac cctgcgggag gtgccggtgg acccgcccct
     3661 gaccaaggtg ttccccgacg acgaatcgga cgacgaatcg gacgacgggg aggacggcga
     3721 cccggactct cggacgttcg tcgcgtacgg ggacgacggc gacctggcgg gcttcgtggt
     3781 cgtctcgtac tccggctgga accgccggct gaccgtcgag gacatcgagg tcgccccgga
     3841 gcaccggggg cacggggtcg ggcgcgcgtt gatggggctc gcgacggagt tcgcccgcga
     3901 gcggggtgcc gggcacctct ggctggaggt caccaacgtc aacgcaccgg cgatccacgc
     3961 gtaccggcgg atggggttca ccctctgcgg cctggacacc gccctgtacg acggcaccgc
     4021 ctcggacggc gagcaggcgc tctacatgag catgccctgc ccctaataat attattttta
     4081 aataatcaaa taatatttca tataatgttg agaatattgt tcaactaaga ttatattatg
     4141 atacaatacc aattaaatta tctctttgag atattaaata ttcaacaact tgataataat
     4201 tatttttaat tatggtatct aaacgatgtt gctccagaaa gttaacactc tcataagata
     4261 tgagagcatc gctcttttag gaattattta ggcggtgaat taaaaagaca atctattatt
     4321 aacaagttaa gcgattttta atacaatcaa attagaatta aaattatgtt gtccataaat
     4381 ctagaatata gtcaagctct aaaaatttgt aaatgaatag tgtccaaaaa tttagaaact
     4441 tgtggtactt aaattagaaa tattttatgt ttgtttacta aattttagcg ttgctaaaat
     4501 tggtgataca aagtccaaat tgtcaattta agaaaatatg attatctcaa aaaaattgaa
     4561 aaaaatatat tcaaaatagt cctttagagc ggccgcagcg ataaggatcc gcgtatggtg
     4621 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     4681 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4741 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4801 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     4861 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4921 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4981 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     5041 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     5101 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     5161 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     5221 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5281 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     5341 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5401 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5461 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5521 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5581 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5641 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5701 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     5761 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5821 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     5881 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5941 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6001 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6061 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6121 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6181 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6241 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6301 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6361 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6421 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6481 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6541 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6601 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6661 ctggcctttt gctcacatgg ctcgac
//
