LOCUS       sequence-467518-        5741 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Encodes V5-GFP with an N-terminal nuclear export sequence and a 
            C-terminal FLAG-TEV cleavage site-AviTag. Used to express a control 
            substrate for ALIBi..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5741)
  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 5741)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_FTA
FEATURES             Location/Qualifiers
     source          1..5741
                     /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..2531)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1818..1820,1821..2531)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /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"
     CDS             2550..2573
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2583..2603
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2619..2663
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     primer_bind     complement(2763..2782)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2828..2883
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2882..2901)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3244..3260)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3244..3260)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3257..3279)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3268..3284
                     /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(3292..3298,3299..3316,3317..3322))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3337..3358
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3417..3612
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3463..3598
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3618..3752
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3655..3674)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3709..3728
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3820..3837)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3991..4579)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4071..4090)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4750..5541,5542..5610))
                     /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     5373..5392
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5611..5715)
                     /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 gggtagtgga tctagcggcg attacaaaga cgatgacgac aagggatctg
     2581 gtgagaacct gtacttccag ggcggttctg gcagtggagg cctgaacgac atcttcgagg
     2641 cccagaagat tgaatggcac gagtgatcga ggatatcgcg gccgcactcc tcaggtgcag
     2701 gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga
     2761 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     2821 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     2881 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     2941 tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga
     3001 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg
     3061 acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta
     3121 aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc
     3181 atagctgtcc ctcttctctt atggagatcc ctcgacctgc agcccaagct tggcgtaatc
     3241 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3301 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3361 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc ggatccgcat
     3421 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3481 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3541 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3601 ggcttttgca aaaagctaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3661 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3721 aactcatcaa tgtatcttat catgtctgga tccgctgcat taatgaatcg gccaacgcgc
     3781 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     3841 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3901 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3961 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4021 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4081 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4141 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4201 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4261 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4321 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4381 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4441 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4501 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4561 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4621 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4681 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4741 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4801 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     4861 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     4921 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     4981 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5041 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     5101 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5161 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5221 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5281 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5341 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5401 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5461 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5521 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5581 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5641 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5701 aataggggtt ccgcgcacat ttccccgaaa agtgccacct g
//
