LOCUS       sequence-373723-        6203 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid for incorporation of O-Me-Tyr into an EGFP reporter for 
            zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6203)
  AUTHORS   Wu KL, Moore JA, Miller MD, Chen Y, Lee C, Xu W, Peng Z, Duan Q, 
            Phillips GN Jr, Uribe RA, Xiao H
  TITLE     Expanding the eukaryotic genetic code with a biosynthesized 21st 
            amino acid.
  JOURNAL   Protein Sci. 2022 Oct;31(10):e4443. doi: 10.1002/pro.4443.
  PUBMED    36173166
REFERENCE   2  (bases 1 to 6203)
  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: "Protein 
            Sci."; date: "2022-10"; volume: "31(10):e4443. doi"; pages: " 
            10.1002/pro.4443"
COMMENT     Sequence Label: pCS2-EcYRS-EGFP*
FEATURES             Location/Qualifiers
     source          1..6203
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        34..52
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     34..51
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1374..1427
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1428..1430,1431..2141)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT*GKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1470..1491)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1731..1750)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2078..2099
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2151..2180
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2196..2213
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2254..2388
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2278..2297)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2332..2351
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2501..2521)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2501..2519)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2540..2556)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2540..2556)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2553..2575)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2564..2580
                     /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(2588..2594,2595..2612,2613..2618))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2633..2654
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2771..2788)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2942..3530)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3022..3041)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3701..4492,4493..4561))
                     /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     4324..4343
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4562..4666)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4692..5147)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4829..4850)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5041..5060
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5218..6202
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 gccattctgc ctggggacgt cggagcaagc ttgatttagg tgacactata gaatacaagc
       61 tacttgttct ttttgcagga tccatggcaa gcagtaactt gattaaacaa ttgcaagagc
      121 gggggctggt agcccaggtg acggacgagg aagcgttagc agagcgactg gcgcaaggcc
      181 cgatcgcact cgtgtgtggc ttcgatccta ccgctgacag cttgcatttg gggcatcttg
      241 ttccattgtt atgcctgaaa cgcttccagc aggcgggcca caagccggtt gcgctggtag
      301 gcggcgcgac gggtctgatt ggcgacccga gcttcaaagc tgccgagcgt aagctgaaca
      361 ccgaagaaac tgttcaggag tgggtggaca aaatccgtaa gcaggttgcc ccgttcctcg
      421 atttcgactg tggagaaaac tctgctatcg cggccaataa ttatgactgg ttcggcaata
      481 tgaatgtgct gaccttcctg cgcgatattg gcaaacactt ctccgttaac cagatgatca
      541 acaaagaagc ggttaagcag cgtctcaacc gtgaagatca ggggatttcg ttcactgagt
      601 tttcctacaa cctgctgcag ggttatagta tggcctgttt gaacaaacag tacggtgtgg
      661 tgctgcaaat tggtggttct gaccagtggg gtaacatcac ttctggtatc gacctgaccc
      721 gtcgtctgca tcagaatcag gtgtttggcc tgaccgttcc gctgatcact aaagcagatg
      781 gcaccaaatt tggtaaaact gaaggcggcg cagtctggtt ggatccgaag aaaaccagcc
      841 cgtacaaatt ctaccagttc tggatcaaca ctgcgcgtgc cgacgtttac cgcttcctga
      901 agttcttcac ctttatgagc attgaagaga tcaacgccct ggaagaagaa gataaaaaca
      961 gcggtaaagc accgcgcgcc cagtatgtac tggcggagca ggtgactcgt ctggttcacg
     1021 gtgaagaagg tttacaggcg gcaaaacgta ttaccgaatg cctgttcagc ggttctttga
     1081 gtgcgctgag tgaagcggac ttcgaacagc tggcgcagga cggcgtaccg atggttgaga
     1141 tggaaaaggg cgcagacctg atgcaggcac tggtcgattc tgaactgcaa ccttcccgtg
     1201 gtcaggcacg taaaactatc gcctccaatg ccatcaccat taacggtgaa aaacagtccg
     1261 atcctgaata cttctttaaa gaagaagatc gtctgtttgg tcgttttacc ttactgcgtc
     1321 gcggtaaaaa gaattactgt ctgatttgct ggaaagggcc cgttgggagt ggggaggggc
     1381 gcggcagtct gcttacctgc ggtgatgttg aggaaaaccc aggaccggtg agcaagggcg
     1441 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1501 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctagggcaag ctgaccctga
     1561 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1621 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1681 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     1741 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1801 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1861 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     1921 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1981 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     2041 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     2101 ccgccgccgg gatcactctc ggcatggacg agctgtacaa ggggcccttc gaacaaaaac
     2161 tcatctcaga agaggatctg aatatgcata ccggtcatca tcaccatcac cattgactcg
     2221 agcctctaga actatagtga gtcgtattac gtagatccag acatgataag atacattgat
     2281 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     2341 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     2401 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta attcgcggcc
     2461 gcggcgccaa tgcattgggc ccggtaccca gcttttgttc cctttagtga gggttaattg
     2521 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     2581 ttccacacaa catacgagcc gggagcataa agtgtaaagc ctggggtgcc taatgagtga
     2641 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2701 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2761 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2821 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2881 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2941 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3001 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3061 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3121 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3181 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3241 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3301 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3361 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3421 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3481 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3541 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3601 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3661 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3721 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3781 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3841 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3901 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3961 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4021 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4081 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4141 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4201 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4261 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4321 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4381 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4441 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4501 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4561 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4621 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4681 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     4741 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     4801 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     4861 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     4921 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     4981 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     5041 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     5101 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt
     5161 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagtc
     5221 gaccgccaat tcaatatggc gtatatggac tcatgccaat tcaatatggt ggatctggac
     5281 ctgtgccaat tcaatatggc gtatatggac tcgtgccaat tcaatatggt ggatctggac
     5341 cccagccaat tcaatatggc ggacttggca ccatgccaat tcaatatggc ggacctggca
     5401 ctgtgccaac tggggagggg tctacttggc acggtgccaa gtttgaggag gggtcttggc
     5461 cctgtgccaa gtccgccata ttgaattggc atggtgccaa taatggcggc catattggct
     5521 atatgccagg atcaatatat aggcaatatc caatatggcc ctatgccaat atggctattg
     5581 gccaggttca atactatgta ttggccctat gccatatagt attccatata tgggttttcc
     5641 tattgacgta gatagcccct cccaatgggc ggtcccatat accatatatg gggcttccta
     5701 ataccgccca tagccactcc cccattgacg tcaatggtct ctatatatgg tctttcctat
     5761 tgacgtcata tgggcggtcc tattgacgta tatggcgcct cccccattga cgtcaattac
     5821 ggtaaatggc ccgcctggct caatgcccat tgacgtcaat aggaccaccc accattgacg
     5881 tcaatgggat ggctcattgc ccattcatat ccgttctcac gccccctatt gacgtcaatg
     5941 acggtaaatg gcccacttgg cagtacatca atatctatta atagtaactt ggcaagtaca
     6001 ttactattgg aagtacgcca gggtacattg gcagtactcc cattgacgtc aatggcggta
     6061 aatggcccgc gatggctgcc aagtacatcc ccattgacgt caatggggag gggcaatgac
     6121 gcaaatgggc gttccattga cgtaaatggg cggtaggcgt gcctaatggg aggtctatat
     6181 aagcaatgct cgtttaggga acc
//
