LOCUS       sequence-391218-        6473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pmg741-NAT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6473)
  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 6473)
  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: vHB47
FEATURES             Location/Qualifiers
     source          1..6473
                     /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             3281..3853
                     /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(4417..4436)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4536..4558
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4596..4614)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4682..4786
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4787..4855,4856..5647)
                     /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(5005..5024)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5818..6406
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6307..6326
                     /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 aaaaagctta aaatattagc aattttatca
     2761 tatattttag ttttattaat aattagtgtg tgacaacttt tacatactct ttttttattt
     2821 ttttttagtg aagtgatata gtaagacatg ttccaataat cggccggatc aggacgatct
     2881 aagaaatcgg gaattacaat gctgtatcct gcgcatttat aacaaaatat attgccacaa
     2941 cttcggcaat gatgtttgcg taaaaatgca ccaaatgttg cattacaacc atgacacgag
     3001 gatactctat gatcctcgac ccaaatatta tttttattac caataacaat agatgttgtt
     3061 gttttttcac tcattatatt attaatataa taatatactt atattgtttg aatagatcta
     3121 acattatctg ttaaaaaaat tcgaaaaaaa attgaaaaaa atatgtattg atatagttaa
     3181 tgatataaaa acgtcattat atcattaata catcgaatat cgacttactt gattcttctg
     3241 acacaacagt ctcgaactta aggctagagc caccgatatc atgggtacca ctcttgacga
     3301 cacggcttac cggtaccgca ccagtgtccc gggggacgcc gaggccatcg aggcactgga
     3361 tgggtccttc accaccgaca ccgtcttccg cgtcaccgcc accggggacg gcttcaccct
     3421 gcgggaggtg ccggtggacc cgcccctgac caaggtgttc cccgacgacg aatcggacga
     3481 cgaatcggac gacggggagg acggcgaccc ggactctcgg acgttcgtcg cgtacgggga
     3541 cgacggcgac ctggcgggct tcgtggtcgt ctcgtactcc ggctggaacc gccggctgac
     3601 cgtcgaggac atcgaggtcg ccccggagca ccgggggcac ggggtcgggc gcgcgttgat
     3661 ggggctcgcg acggagttcg cccgcgagcg gggtgccggg cacctctggc tggaggtcac
     3721 caacgtcaac gcaccggcga tccacgcgta ccggcggatg gggttcaccc tctgcggcct
     3781 ggacaccgcc ctgtacgacg gcaccgcctc ggacggcgag caggcgctct acatgagcat
     3841 gccctgcccc taataatatt atttttaaat aatcaaataa tatttcatat aatgttgaga
     3901 atattgttca actaagatta tattatgata caataccaat taaattatct ctttgagata
     3961 ttaaatattc aacaacttga taataattat ttttaattat ggtatctaaa cgatgttgct
     4021 ccagaaagtt aacactctca taagatatga gagcatcgct cttttaggaa ttatttaggc
     4081 ggtgaattaa aaagacaatc tattattaac aagttaagcg atttttaata caatcaaatt
     4141 agaattaaaa ttatgttgtc cataaatcta gaatatagtc aagctctaaa aatttgtaaa
     4201 tgaatagtgt ccaaaaattt agaaacttgt ggtacttaaa ttagaaatat tttatgtttg
     4261 tttactaaat tttagcgttg ctaaaattgg tgatacaaag tccaaattgt caatttaaga
     4321 aaatatgatt atctcaaaaa aattgaaaaa aatatattca aaatagtcct ttagagcggc
     4381 cgcagcgata aggatccgcg tatggtgcac tctcagtaca atctgctctg atgccgcata
     4441 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4501 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4561 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     4621 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     4681 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4741 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4801 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4861 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4921 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4981 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5041 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5101 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5161 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5221 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5281 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5341 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5401 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5461 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5521 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5581 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5641 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5701 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5761 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5821 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5881 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5941 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6001 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6061 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6121 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6181 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6241 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6301 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6361 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6421 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatggctc gac
//
