LOCUS       sequence-391168-        5667 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTPBF-GFP, Geneticin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5667)
  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 5667)
  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: PAM3
FEATURES             Location/Qualifiers
     source          1..5667
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(553..555,556..558,559..1272)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNDHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(598..619)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(859..878)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1206..1227
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      1501..1956
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1588..1607)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1798..1819
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2073..2433
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2073..2264
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2096..2116)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             2664..3458
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2718..2737)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3328..3347
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3522..3570
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(3611..3630)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3730..3752
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3790..3808)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3876..3980
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3981..4049,4050..4841)
                     /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(4199..4218)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5012..5600
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5501..5520
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatctcttg gatcgggcgc tgaacaggac caacccctcc gtctcggagc aggacgagat
       61 catgtacctc caacagcgcc agcacttctc ggccgttcgg gagtagcgcc aaaaagaaaa
      121 gaaaatagcc tcaaataaca caaatagttt cttagaaagg aaatttgtcg actttgtggt
      181 tcccggcttt ttccaggggc gggaggtacg ttttgtccca cttatttctc agcaccagag
      241 gaggaaagaa ggcaggagca cggccggatc gatagcaggt accgaaccaa gaaaaaccag
      301 ccctattgtc tggttttgtt ctacctttta tttgtttatt tattcggggc ttcttcatct
      361 cgtctcgcct ttacgttttg attgatggct ctgcatcgac gaaaatgcga tcctggtagc
      421 cggtcgataa gaatggggag ccgcattttt aatctttaat aatggcccaa cggtaatatt
      481 ggcgccaacg gagggacgtg cacgtgccga cttgcaagag ctcacgattt gtcttgtgcc
      541 tctagaaatc atatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      601 gtcgagctgg acggcgacgt aaacgaccac aagttcagcg tgtccggcga gggcgagggc
      661 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      721 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      781 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      841 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      901 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
      961 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1021 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1081 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1141 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1201 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1261 ctgtacaagt aaagcggcct cgagaattca cgcgtggtac ctctagagtc gacccaacaa
     1321 caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag
     1381 caagtaaaac ctctacaaat gtggtaaaat ccgataagga tcgatccggg ctggcgtaat
     1441 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     1501 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1561 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1621 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1681 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1741 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1801 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1861 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1921 acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac
     1981 gcatctgtgc ggtatttcac accgcatacg cggatctgcg cagcaccatg gcctgaaata
     2041 acctctgaaa gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat
     2101 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2161 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2221 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     2281 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     2341 tttatttatg cagaggccga ggccgcctcg gcctctaagc tattccagaa gtagtgagga
     2401 ggcttttttg gaggatcaaa cgacgccttg caacaagctg agaaaaaacc aggatcggca
     2461 aggaaggatt ttcaacggaa acgttggcat caccgggtat aaaaaggggc caattttttt
     2521 gttgatttgt tgcgcgaatt cttgctttcg gcatcgaatt caagggagaa ggagtcgatt
     2581 cacacataca acaagatcct aggcttttgc aaaaagcttg attcttctga cacaacagtc
     2641 tcgaacttaa ggctagagcc accatgattg aacaagatgg attgcacgca ggttctccgg
     2701 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     2761 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     2821 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     2881 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     2941 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3001 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3061 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3121 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3181 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3241 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3301 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3361 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3421 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     3481 gaccgaccaa gcgacgccca acctgccatc acgatggccg caataaaata tctttatttt
     3541 cattacatct gtgtgttggt tttttgtgtg aatcgatagc gataaggatc cgcgtatggt
     3601 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     3661 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3721 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3781 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     3841 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3901 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3961 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     4021 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     4081 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     4141 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     4201 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     4261 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4321 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     4381 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     4441 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4501 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     4561 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4621 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4681 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4741 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     4801 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     4861 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4921 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4981 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5041 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5101 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     5161 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5221 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5281 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5341 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5401 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5461 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5521 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5581 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5641 gctggccttt tgctcacatg gctcgac
//
