LOCUS       sequence-391124-        5695 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pEF1-GFP, Geneticin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5695)
  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 5695)
  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: PAM10
FEATURES             Location/Qualifiers
     source          1..5695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(581..583,584..586,587..1300)
                     /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(626..647)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(887..906)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1234..1255
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      1529..1984
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1616..1635)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1826..1847
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2101..2461
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2101..2292
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2124..2144)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             2692..3486
                     /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(2746..2765)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3356..3375
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3550..3598
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(3639..3658)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3758..3780
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3818..3836)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3904..4008
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4009..4077,4078..4869)
                     /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(4227..4246)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5040..5628
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5529..5548
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatctgcga ggggagatgg ttgaagaagc tgtggaagag gactgggcgg tactaccagc
       61 agcgaacatg ccatcgcccc ccgaggcccc atgtcgcgtc agcagggacg gcgtagggcc
      121 aggatgtctc gtaggggagg gaggcggatt gttcgccccc tgagttgcag atgcggatcg
      181 tgtgctgctc atttgcttca ttctatgcaa ttggatccaa ttggaatttg tagattaatc
      241 tgttggcaag gaaccaaggt cgattgggcg ggaccatcga agagaaacct cagggaaaag
      301 gcatttcaga tttcttttat atttcgcacg gggcgatcaa acagcgagaa aaccgacaga
      361 cacgtgactc tggagttcct catgaaattt ctgatgttta tgaattttga ttggctattc
      421 tgataaagag gcaagccgga gtttatcttg gaatattctc aggccacgtc atactccggc
      481 aggggggcac gggggacaaa agcgggaggg cgaagcggcc gtcttttttc tttgcttccg
      541 aggagagcgt agactatacc aacaccacca atccacccat atggtgagca agggcgagga
      601 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acgaccacaa
      661 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
      721 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
      781 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
      841 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
      901 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
      961 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1021 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     1081 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     1141 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     1201 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     1261 cgccgggatc actctcggca tggacgagct gtacaagtaa agcggcctcg agaattcacg
     1321 cgtggtacct ctagagtcga cccaacaaca acaattgcat tcattttatg tttcaggttc
     1381 agggggagat gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcc
     1441 gataaggatc gatccgggct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     1501 acagttgcgc agcctgaatg gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg
     1561 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1621 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1681 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1741 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1801 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1861 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1921 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     1981 atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgcg
     2041 gatctgcgca gcaccatggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt
     2101 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2161 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2221 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2281 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2341 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     2401 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggatcaaacg acgccttgca
     2461 acaagctgag aaaaaaccag gatcggcaag gaaggatttt caacggaaac gttggcatca
     2521 ccgggtataa aaaggggcca atttttttgt tgatttgttg cgcgaattct tgctttcggc
     2581 atcgaattca agggagaagg agtcgattca cacatacaac aagatcctag gcttttgcaa
     2641 aaagcttgat tcttctgaca caacagtctc gaacttaagg ctagagccac catgattgaa
     2701 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     2761 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     2821 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     2881 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     2941 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3001 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3061 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3121 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3181 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     3241 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3301 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3361 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3421 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3481 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     3541 gatggccgca ataaaatatc tttattttca ttacatctgt gtgttggttt tttgtgtgaa
     3601 tcgatagcga taaggatccg cgtatggtgc actctcagta caatctgctc tgatgccgca
     3661 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3721 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3781 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     3841 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     3901 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     3961 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4021 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4081 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     4141 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     4201 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     4261 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     4321 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     4381 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     4441 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     4501 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     4561 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     4621 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     4681 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     4741 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     4801 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     4861 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     4921 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     4981 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5041 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5101 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5161 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5221 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5281 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     5341 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     5401 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5461 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5521 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5581 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5641 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatggc tcgac
//
