LOCUS       sequence-430216-        7244 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of NASP ('s' isofrom) fused at its N-terminus 
            to eGFP with a TEV protease site separating the two. Puromycin 
            resistance is expressed from a downstream IRES..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7244)
  AUTHORS   Apta-Smith MJ, Hernandez-Fernaud JR, Bowman AJ
  TITLE     Evidence for the nuclear import of histones H3.1 and H4 as monomers.
  JOURNAL   EMBO J. 2018 Oct 1;37(19):e98714. doi: 10.15252/embj.201798714. Epub
            2018 Sep 3.
  PUBMED    30177573
REFERENCE   2  (bases 1 to 7244)
  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: "EMBO J."; 
            date: "2018-10-1"; volume: "37(19):e98714. doi"; pages: " 
            10.15252/embj.201798714. Epub 2018 Sep 3"
COMMENT     Sequence Label: pCL1032
FEATURES             Location/Qualifiers
     source          1..7244
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(129..148)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             600..620
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     intron          2048..2277
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2260..2279
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2350..2924,2925..2927,2928..2936)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2517..2534)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2744..2763
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2956..3555
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2956..2975)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3452..3472
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3691..3708)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3697..3921
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3968..3987
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4047..4063)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4047..4063)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4060..4082)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4071..4087
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4095..4101,4102..4119,4120..4125))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4140..4161
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4278..4295)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4449..5037)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4529..4548)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5208..5999,6000..6068))
                     /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     5831..5850
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6069..6173)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6248..6267)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6439..6818
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6819..7022
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6973..6993
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      7061..7070
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7067..7069,7070..7072,7073..7244,1..539)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7112..7133)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
       61 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
      121 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
      181 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
      241 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
      301 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
      361 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
      421 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
      481 tccttaagga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
      541 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc ggcagcggcg
      601 aaaaccttta cttccagggc catatggcca tggagtccac agccactgcc gccgtcgccg
      661 cggagctggt ttctgccgac aaaattgaag atgttcctgc tccttctaca tctgcagata
      721 aagtggagag tctggatgtg gatagtgaag ctaagaaact attgggttta ggacagaaac
      781 atctggtgat gggggatatt ccagcagctg tcaatgcatt ccaggaagca gctagtcttt
      841 taggtaagaa gtatggagag acagctaatg agtgtggaga agccttcttt ttctatggga
      901 aatcacttct ggagttggca agaatggaga atggtgtgtt gggaaacgcc ttggaaggtg
      961 tgcatgtgga agaggaagaa ggagaaaaaa cagaagatga atctctggta gaaaataatg
     1021 ataacataga tgaaactgaa ggctcagaag aggatgataa agaaaatgat aagaccgaag
     1081 aaatgccaaa tgattcagtc cttgaaaaca agtctcttca agaaaatgag gaggaggaga
     1141 ttgggaacct agagcttgcc tgggatatgc tggatttagc aaagatcatt tttaaaaggc
     1201 aagaaacaaa agaagcacag ctttatgctg cccaggcaca tcttaaactc ggagaagtta
     1261 gtgttgaatc tgaaaactat gtgcaagctg tggaggagtt ccagtcctgc cttaacctgc
     1321 aggaacagta cctggaagcc cacgaccgtc tccttgcaga gacccactac cagctgggct
     1381 tggcttatgg gtacaactct cagtatgatg aggcagtggc acagttcagc aaatctattg
     1441 aagtcattga gaacagaatg gctgtactaa acgagcaggt gaaggaggct gaaggatcgt
     1501 ctgctgaata caagaaagaa attgaggaac taaaggaact gctacccgaa attagagaga
     1561 agatagaaga tgcaaaggag tctcagcgta gtgggaatgt agctgaactg gctctgaaag
     1621 ctactctggt ggagagttct acttcaggtt tcactcctgg tggaggaggc tcttcagtct
     1681 ccatgattgc cagtagaaag ccaacagacg gtgcttcctc atcaaattgt gtgactgata
     1741 tttcccacct tgtcagaaag aagaggaaac cagaggaaga gagtccccgg aaagatgatg
     1801 caaagaaagc caaacaagag ccggaggtga acggaggcag tggggatgct gtccccagtg
     1861 gaaatgaagt ttcggaaaac atggaggagg aggctgagaa tcaggctgaa agccgggcag
     1921 cagtggaggg gacagtggag gctggagcta cagttgaaag cactgcatgt taaggatccg
     1981 cggccgcata gataactgat ccagtgtgct ggaattaatt cgctgtctgc gagggccagc
     2041 tgttggggtg agtactccct ctcaaaagcg ggcatgactt ctgcgctaag attgtcagtt
     2101 tccaaaaacg aggaggattt gatattcacc tggcccgcgg tgatgccttt gagggtggcc
     2161 gcgtccatct ggtcagaaaa gacaatcttt ttgttgtcaa gcttgaggtg tggcaggctt
     2221 gagatctggc catacacttg agtgacaatg acatccactt tgcctttctc tccacaggtg
     2281 tccactccca ggtccaactg caggtcgagc atgcatctag ggcggccaat tccgcccctc
     2341 tccccccccc ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata
     2401 aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt
     2461 gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct
     2521 cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc
     2581 ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga
     2641 caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc
     2701 ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt
     2761 attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg
     2821 gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc
     2881 gaaccacggg gacgtggttt tcctttgaaa aacacgatga taagcttgcc acaacccaca
     2941 aggagacgac cttccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     3001 gtcccccggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     3061 accgtcgacc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     3121 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     3181 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     3241 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     3301 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     3361 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     3421 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     3481 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     3541 aagcccggtg cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga
     3601 ccccatggct ccgaccgaag ccacccgggg cggccccgcc gaccccgcac ccgcccccga
     3661 ggcccaccga ctctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat
     3721 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     3781 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     3841 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     3901 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tcgagtgcat
     3961 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     4021 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4081 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4141 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4201 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4261 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4321 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4381 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4441 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4501 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4561 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4621 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4681 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4741 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4801 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4861 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4921 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4981 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5041 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5101 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5161 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5221 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5281 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5341 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5401 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5461 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5521 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5581 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5641 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5701 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5761 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5821 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5881 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5941 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6001 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6061 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6121 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6181 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
     6241 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     6301 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
     6361 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
     6421 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     6481 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     6541 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     6601 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     6661 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     6721 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     6781 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     6841 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     6901 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     6961 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     7021 ctggtttagt gaaccgtcag atccgctagc gctaccggtc gccaccatgg tgagcaaggg
     7081 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     7141 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     7201 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg ccca
//
