LOCUS       sequence-260251-        7429 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Visualization of ERK activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7429)
  AUTHORS   Goedhart, J, Gadella, D.
  TITLE     Goedhart/Gadella plasmids
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 7429)
  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: 
            "unpublished"
COMMENT     Sequence Label: PB-H2A-LSSmOrange2-2A-ERK-KTR-LSS-SGFP2
FEATURES             Location/Qualifiers
     source          1..7429
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   13..47
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        440..975
                     /label=mCMV promoter
                     /note="mouse cytomegalovirus (CMV) immediate early 
                     promoter"
     intron          999..1941
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1898..1918
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2386..2395
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2392..3096
                     /codon_start=1
                     /product="monomeric orange fluorescent protein with a large
                     Stokes shift (Shcherbakova et al., 2012)"
                     /label=LSSmOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALK
                     GEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELY"
     primer_bind     2805..2824
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3115..3171
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     regulatory      3433..3442
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3439..3441,3442..3444,3445..4155)
                     /codon_start=1
                     /product="strongly enhanced green fluorescent protein 
                     (Kremers et al., 2007)"
                     /label=SGFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSIQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3484..3505)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3745..3764)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4092..4113
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(4194..4212)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4195..4212)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4232..4249)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4238..4462
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(4594..5182)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4674..4693)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5257..5391
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5294..5313)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5348..5367
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(5396..5995)
                     /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(5479..5499)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     5976..5995
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     repeat_region   6398..6460
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     CDS             complement(join(6552..7343,7344..7412))
                     /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     7175..7194
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggcgcgcctt aaccctagaa agatagtctg cgtaaaattg acgcatgcat tcttgaaata
       61 ttgctctctc tttctaaata gcgcgaatcc gtcgctgtgc atttaggaca tctcagtcgc
      121 cgcttggagc tcccgtgagg cgtgcttgtc aatgcggtaa gtgtcactga ttttgaacta
      181 taacgaccgc gtgagtcaaa atgacgcatg attatctttt acgtgacttt taagatttaa
      241 ctcatacgat aattatattg ttatttcatg ttctacttac gtgataactt attatatata
      301 tattttcttg ttatagatat catcgataac aggaaagttc cattggagcc aagtacattg
      361 agtcaatagg gactttccaa tgggttttgc ccagtacata aggtcaatgg gaggtaagcc
      421 aatgggtttt tcccattact ggcacgtata ctgagtcatt agggactttc caatgggttt
      481 tgcccagtac ataaggtcaa taggggtgaa tcaacaggaa agttccattg gagccaagta
      541 cactgagtca atagggactt tccattgggt tttgcccagt acaaaaggtc aatagggggt
      601 gagtcaatgg gtttttccca ttattggcac gtacataagg tcaatagggg tgagtcattg
      661 ggtttttcca gccaatttaa ttaaaacgcc atgtactttc ccaccattga cgtcaatggg
      721 ctattgaaac taatgcaacg tgacctttaa acggtacttt cccatagctg attaatggga
      781 aagtaccgtt ctcgagccaa tacacgtcaa tgggaagtga aagggcagcc aaaacgtaac
      841 accgccccgg ttttcccctg gaaattccat attggcacgc attctattgg ctgagctgcg
      901 ttctacgtgg gtataagagg cgcgaccagc gtcggtaccg tcgcagtctt cggtctgacc
      961 accgtagaac gcagagctcc tcgctgcagg caagcttggt aagtgccgtg tgtggttccc
     1021 gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacgcccc
     1081 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
     1141 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
     1201 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
     1261 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
     1321 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
     1381 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
     1441 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
     1501 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     1561 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     1621 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     1681 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     1741 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     1801 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     1861 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     1921 agtttttttc ttccatttca gggatccact agtaacggcc gccagtgtgc tggaattctg
     1981 atgtcgggac gcggcaagca gggaggcaaa gctcgcgcca aagccaagac ccgctcttct
     2041 cgtgccggtc tccagttccc cgtgggccga gtgcaccgac tgctccgcaa gggcaactat
     2101 gctgagcggg tcggggccgg cgcgccggtg tacctggcgg cggtgctgga gtacctgact
     2161 gccgagatcc tggagctggc gggcaacgcc gcccgcgaca acaagaagac ccgcattatc
     2221 ccgcgccact tgcagctggc catccgcaac gacgaggagc tcaacaagct gctgggcaaa
     2281 gtaaccatcg ctcagggtgg tgtcctgccc aacatccagg ctgtgctact gcccaagaag
     2341 accgagagtc accacaaggc caaaggcaaa cgggatccac cggtcgccac catggtgagc
     2401 aagggcgagg agaataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcgcatg
     2461 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     2521 gagggctttc agaccgttaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
     2581 gacatcttgt cccctcagtt cacctacggc tccaaggcct acgtgaagca ccccgccgac
     2641 atccccgact acctcaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     2701 ttcgaggacg gcggcgtggt gaccgtgact caggactcct ccctgcagga cggcgagttc
     2761 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     2821 aagaccatgg gcatggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag
     2881 ggcgaggaca agctcaggct gaagctgaag gacggcggcc actacacctc cgaggtcaag
     2941 accacctaca aggccaagaa gcccgtgcag ttgcccggcg cctacatcgt cgacatcaag
     3001 ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag
     3061 ggccgccact ccaccggcgg catggacgag ctgtaccaaa ccatggggag cggggctacc
     3121 aacttctccc tcctcaagca ggcaggggac gtcgaggaaa atcctggacc catgaaaggc
     3181 cgcaagcctc gcgatctgga gttgcctctc agccctagcc tcctgggggg acagggccct
     3241 gagcgcacac ccgggtccgg cacctctagt ggtttgcagg ctccgggacc tgccctgtca
     3301 ccctccaagc gcagcggcct ggaggatcct gccacaccca gcaaaaaacc aaggaccccc
     3361 tcagtcagct cgcggttaga gcgactgacg ctccagtctt ctttccaatt cccgtccggc
     3421 ggcataccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     3481 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     3541 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     3601 cccgtgccct ggcccaccct cgtgaccacc ctgagctacg gcgtgcagtg cttcgcccgc
     3661 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     3721 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     3781 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     3841 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcacc
     3901 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac
     3961 ggcggcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     4021 ctgctgcccg acaaccacta cctgagcatc cagtccaagc tgagcaaaga ccccaacgag
     4081 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     4141 gacgagctgt acaagtaaag cggccgctcg agcatgcatc tagagggccc tattctatag
     4201 tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     4261 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4321 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4381 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     4441 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagtggcgg taatacggtt
     4501 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4561 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4621 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4681 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4741 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4801 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4861 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4921 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4981 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5041 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5101 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5161 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5221 gtggaacgaa aactcacgtt aagggatttt ggtcataact tgtttattgc agcttataat
     5281 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5341 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgcttcagg
     5401 caccgggctt gcgggtcatg caccaggtgc gcggtccttc gggcacctcg acgtcggcgg
     5461 tgacggtgaa gccgagccgc tcgtagaagg ggaggttgcg gggcgcggag gtctccagga
     5521 aggcgggcac cccggcgcgc tcggccgcct ccactccggg gagcacgacg gcgctgccca
     5581 gacccttgcc ctggtggtcg ggcgagacgc cgacggtggc caggaaccac gcgggctcct
     5641 tgggccggtg cggcgccagg aggccttcca tctgttgctg cgcggccagc ctggaaccgc
     5701 tcaactcggc catgcgcggg ccgatctcgg cgaacaccgc ccccgcttcg acgctctccg
     5761 gcgtggtcca gaccgccacc gcggcgccgt cgtccgcgac ccacaccttg ccgatgtcga
     5821 gcccgacgcg cgtgaggaag agttcttgca gctcggtgac ccgctcgatg tggcggtccg
     5881 ggtcgacggt gtggcgcgtg gcggggtagt cggcgaacgc ggcggcgagg gtgcgtacgg
     5941 cccgggggac gtcgtcgcgg gtggcgaggc gcaccgtggg cttgtactcg gtcatggtgg
     6001 cctgcagagt cgctctgtgt tcgaggccac acgcgtcacc ttaatatgcg aagtggacct
     6061 gggaccgcgc cgccccgact gcatctgcgt gttttcgcca atgacaagac gctgggcggg
     6121 gtttgtgtca tcatagaact aaagacatgc aaatatattt cttccgggga caccgccagc
     6181 aaacgcgagc aacgggccac ggggatgaag cagctggcta gctaaaagtt ttgttacttt
     6241 atagaagaaa ttttgagttt ttgttttttt ttaataaata aataaacata aataaattgt
     6301 ttgttgaatt tattattagt atgtaagtgt aaatataata aaacttaata tctattcaaa
     6361 ttaataaata aacctcgata tacagaccga taaaacacat gcgtcaattt tacgcatgat
     6421 tatctttaac gtacgtcaca atatgattat ctttctaggg ttaattcgaa cagctggttc
     6481 tttccgcctc aggactcttc ctttttcaat aaatcaatct aaagtatata tgagtaaact
     6541 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6601 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6661 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     6721 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6781 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6841 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6901 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6961 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     7021 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7081 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7141 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7201 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7261 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7321 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7381 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaat
//
