LOCUS       sequence-391170-        5427 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTFIID-GFP, Geneticin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5427)
  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 5427)
  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: PAM1
FEATURES             Location/Qualifiers
     source          1..5427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(336..338,339..341,342..1055)
                     /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(381..402)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(642..661)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     989..1010
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      1255..1710
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1342..1361)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1552..1573
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1827..2187
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1827..2018
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1850..1870)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             2418..3212
                     /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(2472..2491)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3082..3101
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3276..3324
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(3365..3384)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3484..3506
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3544..3562)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3630..3734
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3735..3803,3804..4595)
                     /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(3953..3972)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4766..5354
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5255..5274
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac aagtaaagcg gcctcgagaa
      121 ttcacgcgtc cggaggccta aacgacgcct tgcaacaagc tgagaaaaaa ccaggatcgg
      181 caaggaagga ttttcaacgg aaacgttggc atcaccgggt ataaaaaggg gccaattttt
      241 ttgttgattt gttgcgcgaa ttcttgcttt cggcatcgaa ttcaagggag aaggagtcga
      301 ttcacacata caacaagcat atgctagcga tatccatggt gagcaagggc gaggagctgt
      361 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacgac cacaagttca
      421 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
      481 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
      541 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
      601 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
      661 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
      721 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
      781 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
      841 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
      901 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
      961 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     1021 ggatcactct cggcatggac gagctgtaca agtaatctag agtcgaccca acaacaacaa
     1081 ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcaagta
     1141 aaacctctac aaatgtggta aaatccgata aggatcgatc cgggctggcg taatagcgaa
     1201 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc
     1261 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1321 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1381 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1441 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1501 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1561 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1621 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1681 attttaacaa aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct
     1741 gtgcggtatt tcacaccgca tacgcggatc tgcgcagcac catggcctga aataacctct
     1801 gaaagaggaa cttggttagg taccttctga ggcggaaaga accagctgtg gaatgtgtgt
     1861 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     1921 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     1981 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     2041 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     2101 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     2161 tttggaggat caaacgacgc cttgcaacaa gctgagaaaa aaccaggatc ggcaaggaag
     2221 gattttcaac ggaaacgttg gcatcaccgg gtataaaaag gggccaattt ttttgttgat
     2281 ttgttgcgcg aattcttgct ttcggcatcg aattcaaggg agaaggagtc gattcacaca
     2341 tacaacaaga tcctaggctt ttgcaaaaag cttgattctt ctgacacaac agtctcgaac
     2401 ttaaggctag agccaccatg attgaacaag atggattgca cgcaggttct ccggccgctt
     2461 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     2521 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     2581 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     2641 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     2701 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     2761 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     2821 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     2881 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     2941 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     3001 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     3061 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     3121 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     3181 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     3241 ccaagcgacg cccaacctgc catcacgatg gccgcaataa aatatcttta ttttcattac
     3301 atctgtgtgt tggttttttg tgtgaatcga tagcgataag gatccgcgta tggtgcactc
     3361 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     3421 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3481 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     3541 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     3601 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     3661 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     3721 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     3781 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     3841 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     3901 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     3961 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4021 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4081 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4141 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4201 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     4261 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     4321 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     4381 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     4441 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     4501 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     4561 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     4621 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     4681 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     4741 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4801 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4861 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     4921 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4981 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5041 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5101 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5161 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5221 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5281 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5341 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5401 cttttgctca catggctcga cagatct
//
