LOCUS       sequence-391223-        6297 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pneo650-NAT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6297)
  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 6297)
  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: vHB63
FEATURES             Location/Qualifiers
     source          1..6297
                     /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             3365..3937
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     polyA_signal    4152..4200
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4241..4260)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4360..4382
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4420..4438)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4506..4610
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4611..4679,4680..5471)
                     /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(4829..4848)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5642..6230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6131..6150
                     /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 aaaaagcttc ggctctgcag ttgatctctt
     2761 gcgtctgcgg tttttctcct agcgaccttg ttgggccgag ggaaaaaaat agtcgggccg
     2821 ctggtggctc tctgggtcag cgcggagagg aaatagacaa gaaaataaat taaaaaaaga
     2881 ccaaacaaat cgtgccgatg gtgcgagaaa aagggaggcc acaacgcctt ggcctcttct
     2941 ctttcgtttt ccctctgcgc ggtgcggcgc tgggcggggc gcgtgggtcc aaaaaaaaaa
     3001 ggacgacaca cacacacaaa aaatacgaac aaaaaatcta cgacgaatag aaggccccgg
     3061 ttcgaggccg gcccgaaagg aggcacccca atggcgccca acggcggggc acgccgaaac
     3121 gacctgccgt cgccggccaa tgccctaccg taaacaaaaa aagaacacaa gcgtcttcaa
     3181 aaaaagacga ttggttgcat gctttgataa atagatcgga cctcgcatcg tcaaaccgta
     3241 tcgacaacag caaccaacac actctcgaca acaacaacaa caacaataac cgcaaccgca
     3301 accatcatca gcttgattct tctgacacaa cagtctcgaa cttaaggcta gagccaccga
     3361 tatcatgggt accactcttg acgacacggc ttaccggtac cgcaccagtg tcccggggga
     3421 cgccgaggcc atcgaggcac tggatgggtc cttcaccacc gacaccgtct tccgcgtcac
     3481 cgccaccggg gacggcttca ccctgcggga ggtgccggtg gacccgcccc tgaccaaggt
     3541 gttccccgac gacgaatcgg acgacgaatc ggacgacggg gaggacggcg acccggactc
     3601 tcggacgttc gtcgcgtacg gggacgacgg cgacctggcg ggcttcgtgg tcgtctcgta
     3661 ctccggctgg aaccgccggc tgaccgtcga ggacatcgag gtcgccccgg agcaccgggg
     3721 gcacggggtc gggcgcgcgt tgatggggct cgcgacggag ttcgcccgcg agcggggtgc
     3781 cgggcacctc tggctggagg tcaccaacgt caacgcaccg gcgatccacg cgtaccggcg
     3841 gatggggttc accctctgcg gcctggacac cgccctgtac gacggcaccg cctcggacgg
     3901 cgagcaggcg ctctacatga gcatgccctg cccctaataa gcggcgccgt gccgcctgtc
     3961 tgttttctct atctcctttt tgcagttcat gcgtgtggac gcgcaacttt tttcaatgca
     4021 aggaccacaa caggcaataa aaaacataaa aacacaaaat gtctctaagg cacagcggcc
     4081 gcgatatcgc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4141 acgatggccg caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg
     4201 aatcgatagc gataaggatc cgcgtatggt gcactctcag tacaatctgc tctgatgccg
     4261 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4321 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4381 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     4441 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     4501 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4561 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4621 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4681 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4741 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4801 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4861 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4921 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4981 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5041 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5101 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5161 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5221 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     5281 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     5341 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     5401 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     5461 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     5521 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5581 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5641 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5701 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5761 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5821 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5881 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5941 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6001 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6061 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6121 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6181 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6241 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg gctcgac
//
