LOCUS       sequence-467509-        5609 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes V5-GFP with an N-terminal nuclear export sequence. Used as a
            no-ALIBi negative control..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5609)
  AUTHORS   Zhang Z, Xu A, Bai Y, Chen Y, Cates K, Kerr C, Bermudez A, Susanto 
            TT, Wysong K, Garcia Marques FJ, Nolan GP, Pitteri S, Barna M
  TITLE     A subcellular map of translational machinery composition and 
            regulation at the single-molecule level.
  JOURNAL   Science. 2025 Mar 7;387(6738):eadn2623. doi: 
            10.1126/science.adn2623. Epub 2025 Mar 7.
  PUBMED    40048539
REFERENCE   2  (bases 1 to 5609)
  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: "Science."; 
            date: "2025-03-7"; volume: "387(6738):eadn2623. doi"; pages: " 
            10.1126/science.adn2623. Epub 2025 Mar 7"
COMMENT     Sequence Label: pCAGEN_NES_V5_GFP
FEATURES             Location/Qualifiers
     source          1..5609
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1776..1817
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             join(1818..1820,1821..2534)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1818..1820,1821..2531)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1860..1881)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2121..2140)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2468..2489
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2631..2650)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2696..2751
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2750..2769)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3112..3128)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3112..3128)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3125..3147)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3136..3152
                     /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(3160..3166,3167..3184,3185..3190))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3205..3226
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3285..3480
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3331..3466
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3486..3620
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3523..3542)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3577..3596
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3688..3705)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3859..4447)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3939..3958)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4618..5409,5410..5478))
                     /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     5241..5260
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5479..5583)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggggcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tccatgctgc
     1741 agaagaagct ggaggagctg gaactgggct cttccggtaa gcctatccct aaccctctcc
     1801 tcggtctcga ttctacggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     1861 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     1921 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     1981 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     2041 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     2101 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     2161 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     2221 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     2281 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     2341 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     2401 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     2461 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     2521 agctgtacaa gtgatcgagg atatcgcggc cgcactcctc aggtgcaggc tgcctatcag
     2581 aaggtggtgg ctggtgtggc caatgccctg gctcacaaat accactgaga tctttttccc
     2641 tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa
     2701 aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg
     2761 acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca
     2821 acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat
     2881 atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag
     2941 atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta
     3001 catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct
     3061 cttctcttat ggagatccct cgacctgcag cccaagcttg gcgtaatcat ggtcatagct
     3121 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     3181 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3241 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca
     3301 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3361 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3421 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3481 aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3541 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3601 tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3661 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3721 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3781 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3841 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3901 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3961 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4021 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4081 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4141 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4201 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4261 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4321 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4381 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4441 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4501 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4561 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4621 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4681 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4741 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4801 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4861 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4921 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4981 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5041 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5101 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5161 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5221 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5281 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5341 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5401 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5461 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5521 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5581 gcgcacattt ccccgaaaag tgccacctg
//
