LOCUS       sequence-381999-        6926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3xFLAG endogenous tagging of pandoravirus/mollivirus genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6926)
  AUTHORS   Bisio H, Legendre M, Giry C, Philippe N, Alempic JM, Jeudy S, 
            Abergel C
  TITLE     Evolution of giant pandoravirus revealed by CRISPR/Cas9.
  JOURNAL   Nat Commun. 2023 Jan 26;14(1):428. doi: 10.1038/s41467-023-36145-4.
  PUBMED    36702819
REFERENCE   2  (bases 1 to 6926)
  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 
            Commun."; date: "2023-01-26"; volume: "14(1):428. doi"; pages: " 
            10.1038/s41467-023-36145-4"
COMMENT     Sequence Label: vHB179
FEATURES             Location/Qualifiers
     source          1..6926
                     /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             2671..2736
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3994..4566
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     polyA_signal    4781..4829
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4870..4889)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4989..5011
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5049..5067)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5135..5239
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5240..5308,5309..6100)
                     /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(5458..5477)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6271..6859
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6760..6779
                     /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 ccatatgtca gactacaaag accatgacgg tgattataaa
     2701 gatcatgaca tcgactacaa ggatgacgat gacaagtaat cttgttctta ttttctcaat
     2761 aggaaaagaa gacgggatta ttgctttacc tataattata gcgcccgaac taagcgcccg
     2821 gaaaaaggct tagttgacga ggatggaggt tatcgaattt tcggcggatg cctcccggct
     2881 gagtgtgcag atcacagccg taaggatttc ttcaaaccaa gggggtgact ccttgaacaa
     2941 agagaaatca catgatcttc caaaaaacat gtaggagggg acgattgaaa gtccccttga
     3001 aatttgactt tcttcgtctc cttttacaat cttaggagga agaaaaataa ttctctttgc
     3061 ctcgtctttt tctgtctgtc tttgtgccaa aaactcgtgt tccgtttttt cttgtgcgcg
     3121 gtcgctctcg ggccgatccg cagaggcagc gcgagtcgcg caataatcaa aaaaaaaaga
     3181 aggagaacaa gaaaaaaaag cggcaacgta gcgcacaaac tggatttttt atgattgagc
     3241 acaagccgca tgagactttt tgatcgcaag gcagcattgg acgggacgat gtcgccgcca
     3301 ccagggcgac gggagtcgcc caaaaatcgg caaacgagga cggcccgacg ggagcgagcc
     3361 tcaagcttcg gctctgcagt tgatctcttg cgtctgcggt ttttctccta gcgaccttgt
     3421 tgggccgagg gaaaaaaata gtcgggccgc tggtggctct ctgggtcagc gcggagagga
     3481 aatagacaag aaaataaatt aaaaaaagac caaacaaatc gtgccgatgg tgcgagaaaa
     3541 agggaggcca caacgccttg gcctcttctc tttcgttttc cctctgcgcg gtgcggcgct
     3601 gggcggggcg cgtgggtcca aaaaaaaaag gacgacacac acacacaaaa aatacgaaca
     3661 aaaaatctac gacgaataga aggccccggt tcgaggccgg cccgaaagga ggcaccccaa
     3721 tggcgcccaa cggcggggca cgccgaaacg acctgccgtc gccggccaat gccctaccgt
     3781 aaacaaaaaa agaacacaag cgtcttcaaa aaaagacgat tggttgcatg ctttgataaa
     3841 tagatcggac ctcgcatcgt caaaccgtat cgacaacagc aaccaacaca ctctcgacaa
     3901 caacaacaac aacaataacc gcaaccgcaa ccatcatcag cttgattctt ctgacacaac
     3961 agtctcgaac ttaaggctag agccaccgat atcatgggta ccactcttga cgacacggct
     4021 taccggtacc gcaccagtgt cccgggggac gccgaggcca tcgaggcact ggatgggtcc
     4081 ttcaccaccg acaccgtctt ccgcgtcacc gccaccgggg acggcttcac cctgcgggag
     4141 gtgccggtgg acccgcccct gaccaaggtg ttccccgacg acgaatcgga cgacgaatcg
     4201 gacgacgggg aggacggcga cccggactct cggacgttcg tcgcgtacgg ggacgacggc
     4261 gacctggcgg gcttcgtggt cgtctcgtac tccggctgga accgccggct gaccgtcgag
     4321 gacatcgagg tcgccccgga gcaccggggg cacggggtcg ggcgcgcgtt gatggggctc
     4381 gcgacggagt tcgcccgcga gcggggtgcc gggcacctct ggctggaggt caccaacgtc
     4441 aacgcaccgg cgatccacgc gtaccggcgg atggggttca ccctctgcgg cctggacacc
     4501 gccctgtacg acggcaccgc ctcggacggc gagcaggcgc tctacatgag catgccctgc
     4561 ccctaataag cggcgccgtg ccgcctgtct gttttctcta tctccttttt gcagttcatg
     4621 cgtgtggacg cgcaactttt ttcaatgcaa ggaccacaac aggcaataaa aaacataaaa
     4681 acacaaaatg tctctaaggc acagcggccg cgatatcgcg ggactctggg gttcgaaatg
     4741 accgaccaag cgacgcccaa cctgccatca cgatggccgc aataaaatat ctttattttc
     4801 attacatctg tgtgttggtt ttttgtgtga atcgatagcg ataaggatcc gcgtatggtg
     4861 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     4921 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4981 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     5041 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     5101 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     5161 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     5221 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     5281 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     5341 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     5401 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     5461 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5521 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     5581 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5641 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5701 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5761 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5821 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5881 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5941 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6001 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6061 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6121 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6181 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6241 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6301 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6361 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6421 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6481 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6541 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6601 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6661 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6721 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6781 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6841 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6901 ctggcctttt gctcacatgg ctcgac
//
