LOCUS       sequence-385354-        3716 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway p3E 2A fluorophore.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3716)
  AUTHORS   Kemmler CL, Moran HR, Murray BF, Scoresby A, Klem JR, Eckert RL, 
            Lepovsky E, Bertho S, Nieuwenhuize S, Burger S, D'Agati G, Betz C, 
            Puller AC, Felker A, Ditrychova K, Botschi S, Affolter M, Rohner N, 
            Lovely CB, Kwan KM, Burger A, Mosimann C
  TITLE     Next-generation plasmids for transgenesis in zebrafish and beyond.
  JOURNAL   Development. 2023 Mar 28:dev.201531. doi: 10.1242/dev.201531.
  PUBMED    36975217
REFERENCE   2  (bases 1 to 3716)
  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: 
            "Development. 2023 Mar 28:dev.201531. doi: 10.1242/dev.201531."
COMMENT     Sequence Label: pAF020 p3E-2A-mCerulean
FEATURES             Location/Qualifiers
     source          1..3716
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(61..184)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             196..252
                     /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"
     CDS             join(262..264,265..267,268..981)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(307..328)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(568..587)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     915..936
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1018..1036)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1041..1175
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1065..1084)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1119..1138
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(1294..1389)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1422..1440)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1445..1461)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1445..1461)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1563..1582)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1574..2383
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1641..1660)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2530..3118
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3019..3038
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3272..3289
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3448..3475)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3567..3653)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(3702..3716,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(3716,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aaaattctac aggtcactaa taccatctaa gtagttggtt
       61 catagtgact gcatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc
      121 taatttaata tattgatatt tatatcattt tacgtttctc gttcaacttt cttgtacaaa
      181 gtggggggat ctggagccac gaacttctct ctgttaaagc aagcaggaga cgtggaagaa
      241 aaccccggtc ctagggatcc catggtgagc aagggcgagg agctgttcac cggggtggtg
      301 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      361 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
      421 ctgcccgtgc cctggcccac cctcgtgacc accctgacct ggggcgtgca gtgcttcgcc
      481 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
      541 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
      601 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
      661 gacggcaaca tcctggggca caagctggag tacaacgcca tcagcgacaa cgtctatatc
      721 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag
      781 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
      841 gtgctgctgc ccgacaacca ctacctgagc acccagtcca agctgagcaa agaccccaac
      901 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
      961 atggacgagc tgtacaagta agaattcaag gcctctcgag cctctagatt ctgcagccct
     1021 atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag tttggacaaa
     1081 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     1141 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     1201 tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cgcggccgcg gcgccaatgc
     1261 attgggcccg gtaccgatcc accggatctt gaccaacttt attatacaaa gttggcatta
     1321 taaaaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa
     1381 tcattatttg gagctccatg gtagcgttaa cgcggccatc ccctatagtg agtcgtatta
     1441 catggtcata gctgtttcct ggcagctctg gcccgtgtct caaaatctct gatgttacat
     1501 tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa
     1561 tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa
     1621 catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc
     1681 gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa
     1741 aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt
     1801 tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac
     1861 cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga
     1921 aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa
     1981 ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa
     2041 cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt
     2101 ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga
     2161 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg
     2221 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga
     2281 gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat
     2341 gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg
     2401 gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcgcaagct catgaccaaa
     2461 atcccttaac gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     2521 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     2581 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     2641 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     2701 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     2761 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     2821 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     2881 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     2941 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3001 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3061 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3121 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     3181 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     3241 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     3301 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     3361 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
     3421 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
     3481 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
     3541 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
     3601 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
     3661 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgtt
//
