LOCUS       sequence-391169-        5858 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pGAPDH-GFP, Geneticin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5858)
  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 5858)
  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: PAM2
FEATURES             Location/Qualifiers
     source          1..5858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(744..746,747..749,750..1463)
                     /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(789..810)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1050..1069)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1397..1418
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      1692..2147
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1779..1798)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1989..2010
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2264..2624
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2264..2455
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2287..2307)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             2855..3649
                     /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(2909..2928)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3519..3538
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3713..3761
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(3802..3821)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3921..3943
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3981..3999)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4067..4171
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4172..4240,4241..5032)
                     /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(4390..4409)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5203..5791
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5692..5711
                     /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 catatggtga gcaagggcga ggagctgttc accggggtgg
      781 tgcccatcct ggtcgagctg gacggcgacg taaacgacca caagttcagc gtgtccggcg
      841 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
      901 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
      961 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1021 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1081 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1141 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     1201 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     1261 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     1321 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     1381 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     1441 gcatggacga gctgtacaag taaagcggcc tcgagaattc acgcgtggta cctctagagt
     1501 cgacccaaca acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag
     1561 gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tccgataagg atcgatccgg
     1621 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     1681 atggcgaatg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1741 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1801 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1861 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1921 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1981 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2041 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2101 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttcct gatgcggtat
     2161 tttctcctta cgcatctgtg cggtatttca caccgcatac gcggatctgc gcagcaccat
     2221 ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc
     2281 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2341 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     2401 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     2461 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     2521 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     2581 agtagtgagg aggctttttt ggaggatcaa acgacgcctt gcaacaagct gagaaaaaac
     2641 caggatcggc aaggaaggat tttcaacgga aacgttggca tcaccgggta taaaaagggg
     2701 ccaatttttt tgttgatttg ttgcgcgaat tcttgctttc ggcatcgaat tcaagggaga
     2761 aggagtcgat tcacacatac aacaagatcc taggcttttg caaaaagctt gattcttctg
     2821 acacaacagt ctcgaactta aggctagagc caccatgatt gaacaagatg gattgcacgc
     2881 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     2941 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3001 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     3061 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     3121 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     3181 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     3241 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     3301 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     3361 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     3421 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     3481 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     3541 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     3601 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     3661 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgatggcc gcaataaaat
     3721 atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag cgataaggat
     3781 ccgcgtatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     3841 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3901 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3961 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4021 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4081 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4141 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4201 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4261 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4321 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4381 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4441 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4501 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4561 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4621 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4681 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4741 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4801 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4861 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     4921 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4981 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5041 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5101 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5161 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5221 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5281 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5341 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5401 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5461 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5521 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5581 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5641 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5701 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5761 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5821 cctggccttt tgctggcctt ttgctcacat ggctcgac
//
