LOCUS       sequence-375799-        6271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MCP-EGFP in mammalian cells. To clone this plasmid, 
            Addgene plasmid #119908 was used. We replaced the tandem coat 
            proteins and double tagRFP with a single coat protein fused to 
            EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6271)
  AUTHORS   Brouwer I, de Kort MAC, Lenstra TL
  TITLE     Measuring Transcription Dynamics of Individual Genes Inside Living 
            Cells.
  JOURNAL   Methods Mol Biol. 2024;2694:235-265. doi: 
            10.1007/978-1-0716-3377-9_12.
  PUBMED    37824008
REFERENCE   2  (bases 1 to 6271)
  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: "Methods Mol
            Biol."; date: "2024"; volume: "2694:235-265. doi"; pages: " 
            10.1007/978-1-0716-3377-9_12"
COMMENT     Sequence Label: MCP-EGFP-PiggyBac
FEATURES             Location/Qualifiers
     source          1..6271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        103..1314
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     1151..1170
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     protein_bind    1331..1355
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1406..1426
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1430..1456
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1430..1456
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1457..1468
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(1845..1869)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1871..1873,1874..2587)
                     /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(1871..1873,1874..2584)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1913..1934)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2174..2193)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2521..2542
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2609..2633)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    2749..2870
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2786..2805)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2840..2859
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3056..3118
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3153..3172)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3154..3172)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3182..3199)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3182..3198)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3191..3213)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3340..3795
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3427..3446)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3637..3658
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3821..3925
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3926..3994,3995..4786)
                     /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(4144..4163)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4957..5545
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5446..5465
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5699..5716
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5833..5854
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5869..5874,5875..5892,5893..5899)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5907..5923
                     /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)."
     primer_bind     5912..5934
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5931..5947
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5931..5947
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5966..5986
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5968..5986
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   6034..6068
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 aattatattg ttatttcgtg ttctacttac gtgataactt attatatata tattttcttg
       61 ttatagatat ccttctcgac attgattatt gactagagat ctggcctccg cgccgggttt
      121 tggcgcctcc cgcgggcgcc cccctcctca cggcgagcgc tgccacgtca gacgaagggc
      181 gcagcgagcg tcctgatcct tccgcccgga cgctcaggac agcggcccgc tgctcataag
      241 actcggcctt agaaccccag tatcagcaga aggacatttt aggacgggac ttgggtgact
      301 ctagggcact ggttttcttt ccagagagcg gaacaggcga ggaaaagtag tcccttctcg
      361 gcgattctgc ggagggatct ccgtggggcg gtgaacgccg atgattatat aaggacgcgc
      421 cgggtgtggc acagctagtt ccgtcgcagc cgggatttgg gtcgcggttc ttgtttgtgg
      481 atcgctgtga tcgtcacttg gtgagtagcg ggctgctggg ctggccgggg ctttcgtggc
      541 cgccgggccg ctcggtggga cggaagcgtg tggagagacc gccaagggct gtagtctggg
      601 tccgcgagca aggttgccct gaactggggg ttggggggag cgcagcaaaa tggcggctgt
      661 tcccgagtct tgaatggaag acgcttgtga ggcgggctgt gaggtcgttg aaacaaggtg
      721 gggggcatgg tgggcggcaa gaacccaagg tcttgaggcc ttcgctaatg cgggaaagct
      781 cttattcggg tgagatgggc tggggcacca tctggggacc ctgacgtgaa gtttgtcact
      841 gactggagaa ctcggtttgt cgtctgttgc gggggcggca gttatggcgg tgccgttggg
      901 cagtgcaccc gtacctttgg gagcgcgcgc cctcgtcgtg tcgtgacgtc acccgttctg
      961 ttggcttata atgcagggtg gggccacctg ccggtaggtg tgcggtaggc ttttctccgt
     1021 cgcaggacgc agggttcggg cctagggtag gctctcctga atcgacaggc gccggacctc
     1081 tggtgagggg agggataagt gaggcgtcag tttctttggt cggttttatg tacctatctt
     1141 cttaagtagc tgaagctccg gttttgaact atgcgctcgg ggttggcgag tgtgttttgt
     1201 gaagtttttt aggcaccttt tgaaatgtaa tcatttgggt caatatgtaa ttttcagtgt
     1261 tagactagta aattgtccgc taaattctgg ccgtttttgg cttttttgtt agacgaagct
     1321 cgaggatatc acaagtttgt acaaaaaagc aggctttaaa ggaaccaatt cagtcgactg
     1381 gatccggtac cgaattcgca tgggcccaaa aaagaaaaga aaagttggct acccctacga
     1441 cgtgcccgac tacgccatcg aaggccgcca tatgctagcc gttaaaatgg cttctaactt
     1501 tactcagttc gttctcgtcg acaatggcgg aactggcgac gtgactgtcg ccccaagcaa
     1561 cttcgctaac gggatcgctg aatggatcag ctctaactcg cgttcacagg cttacaaagt
     1621 aacctgtagc gttcgtcaga gctctgcgca gaatcgcaaa tacaccatca aagtcgaggt
     1681 gcctaaaggc gcctggcgtt cgtacttaaa tatggaacta accattccaa ttttcgccac
     1741 gaattccgac tgcgagctta ttgttaaggc aatgcaaggt ctcctaaaag atggaaaccc
     1801 gattccctca gcaatcgcag caaactccgg catctacgcg gattcttcaa ctttcttgta
     1861 caaagtgggc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     1921 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     1981 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     2041 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     2101 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     2161 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     2221 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     2281 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     2341 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     2401 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     2461 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     2521 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     2581 caagtaaggc cgcactcgag atatctagac ccagctttct tgtacaaagt ggtgcgactc
     2641 tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     2701 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     2761 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     2821 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aggaattcga
     2881 taaaagtttt gttactttat agaagaaatt ttgagttttt gttttttttt aataaataaa
     2941 taaacataaa taaattgttt gttgaattta ttattagtat gtaagtgtaa atataataaa
     3001 acttaatatc tattcaaatt aataaataaa cctcgatata cagaccgata aaacacatgc
     3061 gtcaatttta cgcatgatta tctttaacgt acgtcacaat atgattatct ttctagggtt
     3121 aatctagctg cggcgcgccg gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
     3181 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     3241 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     3301 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     3361 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3421 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3481 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3541 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3601 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3661 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3721 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     3781 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3841 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3901 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     3961 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     4021 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     4081 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     4141 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     4201 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     4261 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     4321 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     4381 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     4441 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     4501 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     4561 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     4621 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     4681 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     4741 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     4801 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4861 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4921 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4981 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5041 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     5101 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5161 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5221 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5281 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5341 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5401 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5461 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5521 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5581 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5641 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     5701 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     5761 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     5821 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     5881 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     5941 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga
     6001 gctccaccgc ggggcgcgcc gcagctagat taaccctaga aagatagtct gcgtaaaatt
     6061 gacgcatgca ttcttgaaat attgctctct ctttctaaat agcgcgaatc cgtcgctgtg
     6121 catttaggac atctcagtcg ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta
     6181 agtgtcactg attttgaact ataacaaccg cgtgagtcaa aatgacgcat gattatcttt
     6241 tacgtgactt ttaagattta actcatacga t
//
