LOCUS       sequence-391171-        5648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pGAPDH-GFP, Nourseothricin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5648)
  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 5648)
  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: PAM24
FEATURES             Location/Qualifiers
     source          1..5648
                     /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             2861..3433
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     polyA_signal    3503..3551
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(3592..3611)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3711..3733
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3771..3789)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3857..3961
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3962..4030,4031..4822)
                     /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(4180..4199)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4993..5581
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5482..5501
                     /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 caccgatatc atgggtacca ctcttgacga
     2881 cacggcttac cggtaccgca ccagtgtccc gggggacgcc gaggccatcg aggcactgga
     2941 tgggtccttc accaccgaca ccgtcttccg cgtcaccgcc accggggacg gcttcaccct
     3001 gcgggaggtg ccggtggacc cgcccctgac caaggtgttc cccgacgacg aatcggacga
     3061 cgaatcggac gacggggagg acggcgaccc ggactctcgg acgttcgtcg cgtacgggga
     3121 cgacggcgac ctggcgggct tcgtggtcgt ctcgtactcc ggctggaacc gccggctgac
     3181 cgtcgaggac atcgaggtcg ccccggagca ccgggggcac ggggtcgggc gcgcgttgat
     3241 ggggctcgcg acggagttcg cccgcgagcg gggtgccggg cacctctggc tggaggtcac
     3301 caacgtcaac gcaccggcga tccacgcgta ccggcggatg gggttcaccc tctgcggcct
     3361 ggacaccgcc ctgtacgacg gcaccgcctc ggacggcgag caggcgctct acatgagcat
     3421 gccctgcccc taagatatcg cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     3481 aacctgccat cacgatggcc gcaataaaat atctttattt tcattacatc tgtgtgttgg
     3541 ttttttgtgt gaatcgatag cgataaggat ccgcgtatgg tgcactctca gtacaatctg
     3601 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     3661 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     3721 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     3781 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     3841 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3901 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3961 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4021 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4081 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4141 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4201 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4261 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4321 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4381 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4441 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4501 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4561 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4621 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4681 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4741 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4801 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4861 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4921 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4981 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5041 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5101 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     5161 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5221 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5281 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5341 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5401 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5461 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5521 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5581 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5641 ggctcgac
//
