LOCUS       sequence-373722-        7397 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid for incorporation of O-Me-Tyr into an EGFP reporter 
            without O-Me-Tyr feeding for zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7397)
  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 7397)
  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-MfnG-EcYRS-EGFP*
FEATURES             Location/Qualifiers
     source          1..7397
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1222..1278
                     /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             2569..2622
                     /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(2623..2625,2626..3336)
                     /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(2665..2686)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2926..2945)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3273..3294
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3346..3375
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3391..3408
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    3449..3583
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3473..3492)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3527..3546
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3696..3716)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3696..3714)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3735..3751)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3735..3751)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3748..3770)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3759..3775
                     /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(3783..3789,3790..3807,3808..3813))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3828..3849
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3966..3983)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4137..4725)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4217..4236)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4896..5687,5688..5756))
                     /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     5519..5538
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5757..5861)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5887..6342)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6024..6045)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6236..6255
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6413..7397
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atccatggtg actccagagg ggaatgtgtc attggtggat
      121 gagagcttgt tagtcggcgt tacggatgag gatcgcgcag tccgttctgc ccaccaattc
      181 tatgagcgtt taattggttt gtgggcgcct gcagtaatgg aggctgccca tgagttgggc
      241 gtttttgctg ccttagcaga agcgcctgct gactcagggg aactggctcg ccgcctggat
      301 tgcgacgccc gtgctatgcg tgtattgctg gacgcattat acgcatatga cgtcattgac
      361 cgcatccatg atactaacgg ctttcgttat ttacttagtg ccgaggcacg tgaatgcctt
      421 cttccgggta cgctgttttc cttagtcggc aagttcatgc atgacatcaa cgtggcctgg
      481 cctgcctggc gcaatcttgc tgaagtggtg cgccacggcg cgcgtgatac gtccggcgcc
      541 gaatctccaa atggcatcgc acaggaagac tatgaatctt tggtcggagg gatcaatttt
      601 tgggcacctc caatcgtgac gaccttgtct cgcaaactgc gtgcctctgg acgttcgggg
      661 gatgcaacag cctccgtgct ggatgttggc tgcggtacgg gactgtactc acagctgctg
      721 ttacgcgagt tccctcgttg gacagccaca ggcttagacg tcgaacgtat cgcgacttta
      781 gctaatgctc aggcactgcg cttgggagtc gaagagcgtt ttgcaactcg tgccggggat
      841 ttctggcgcg gaggctgggg aacaggctac gacttagtct tgtttgccaa tattttccat
      901 ttgcaaacgc cagcatcagc agtccgtctt atgcgtcatg ccgccgcttg tctggcgccc
      961 gacggtttag tggctgtagt agaccaaatt gtagatgccg accgtgaacc caaaacccca
     1021 caggatcgtt ttgctttatt atttgcggcc tcaatgacca acactggtgg cggcgacgca
     1081 tatacattcc aagagtatga agaatggttt accgcagcgg gattgcagcg cattgaaact
     1141 ttggacactc caatgcatcg cattctttta gcccgtcgcg ccaccgaacc tagcgcagtc
     1201 ccggagggac aggggagtgg ggcgaccaat ttcagcctcc ttaaacaggc tggcgatgtg
     1261 gaagagaatc ctggccccat ggcaagcagt aacttgatta aacaattgca agagcggggg
     1321 ctggtagccc aggtgacgga cgaggaagcg ttagcagagc gactggcgca aggcccgatc
     1381 gcactcgtgt gtggcttcga tcctaccgct gacagcttgc atttggggca tcttgttcca
     1441 ttgttatgcc tgaaacgctt ccagcaggcg ggccacaagc cggttgcgct ggtaggcggc
     1501 gcgacgggtc tgattggcga cccgagcttc aaagctgccg agcgtaagct gaacaccgaa
     1561 gaaactgttc aggagtgggt ggacaaaatc cgtaagcagg ttgccccgtt cctcgatttc
     1621 gactgtggag aaaactctgc tatcgcggcc aataattatg actggttcgg caatatgaat
     1681 gtgctgacct tcctgcgcga tattggcaaa cacttctccg ttaaccagat gatcaacaaa
     1741 gaagcggtta agcagcgtct caaccgtgaa gatcagggga tttcgttcac tgagttttcc
     1801 tacaacctgc tgcagggtta tagtatggcc tgtttgaaca aacagtacgg tgtggtgctg
     1861 caaattggtg gttctgacca gtggggtaac atcacttctg gtatcgacct gacccgtcgt
     1921 ctgcatcaga atcaggtgtt tggcctgacc gttccgctga tcactaaagc agatggcacc
     1981 aaatttggta aaactgaagg cggcgcagtc tggttggatc cgaagaaaac cagcccgtac
     2041 aaattctacc agttctggat caacactgcg cgtgccgacg tttaccgctt cctgaagttc
     2101 ttcaccttta tgagcattga agagatcaac gccctggaag aagaagataa aaacagcggt
     2161 aaagcaccgc gcgcccagta tgtactggcg gagcaggtga ctcgtctggt tcacggtgaa
     2221 gaaggtttac aggcggcaaa acgtattacc gaatgcctgt tcagcggttc tttgagtgcg
     2281 ctgagtgaag cggacttcga acagctggcg caggacggcg taccgatggt tgagatggaa
     2341 aagggcgcag acctgatgca ggcactggtc gattctgaac tgcaaccttc ccgtggtcag
     2401 gcacgtaaaa ctatcgcctc caatgccatc accattaacg gtgaaaaaca gtccgatcct
     2461 gaatacttct ttaaagaaga agatcgtctg tttggtcgtt ttaccttact gcgtcgcggt
     2521 aaaaagaatt actgtctgat ttgctggaaa gggcccgttg ggagtgggga ggggcgcggc
     2581 agtctgctta cctgcggtga tgttgaggaa aacccaggac cggtgagcaa gggcgaggag
     2641 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2701 ttcagcgtgt ccggcgaggg cgagggcgat gccacctagg gcaagctgac cctgaagttc
     2761 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     2821 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2881 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2941 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     3001 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     3061 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     3121 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     3181 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     3241 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     3301 gccgggatca ctctcggcat ggacgagctg tacaaggggc ccttcgaaca aaaactcatc
     3361 tcagaagagg atctgaatat gcataccggt catcatcacc atcaccattg actcgagcct
     3421 ctagaactat agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt
     3481 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     3541 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     3601 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaattcg cggccgcggc
     3661 gccaatgcat tgggcccggt acccagcttt tgttcccttt agtgagggtt aattgcgcgc
     3721 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3781 cacaacatac gagccgggag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3841 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3901 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3961 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4021 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4081 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4141 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4201 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4261 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4321 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4381 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4441 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4501 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4561 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4621 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4681 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4741 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4801 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4861 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4921 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4981 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5041 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5101 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5161 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5221 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5281 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5341 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5401 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5461 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5521 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5581 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5641 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5701 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5761 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5821 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5881 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     5941 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     6001 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     6061 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     6121 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     6181 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     6241 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     6301 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
     6361 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagtcgaccg
     6421 ccaattcaat atggcgtata tggactcatg ccaattcaat atggtggatc tggacctgtg
     6481 ccaattcaat atggcgtata tggactcgtg ccaattcaat atggtggatc tggaccccag
     6541 ccaattcaat atggcggact tggcaccatg ccaattcaat atggcggacc tggcactgtg
     6601 ccaactgggg aggggtctac ttggcacggt gccaagtttg aggaggggtc ttggccctgt
     6661 gccaagtccg ccatattgaa ttggcatggt gccaataatg gcggccatat tggctatatg
     6721 ccaggatcaa tatataggca atatccaata tggccctatg ccaatatggc tattggccag
     6781 gttcaatact atgtattggc cctatgccat atagtattcc atatatgggt tttcctattg
     6841 acgtagatag cccctcccaa tgggcggtcc catataccat atatggggct tcctaatacc
     6901 gcccatagcc actcccccat tgacgtcaat ggtctctata tatggtcttt cctattgacg
     6961 tcatatgggc ggtcctattg acgtatatgg cgcctccccc attgacgtca attacggtaa
     7021 atggcccgcc tggctcaatg cccattgacg tcaataggac cacccaccat tgacgtcaat
     7081 gggatggctc attgcccatt catatccgtt ctcacgcccc ctattgacgt caatgacggt
     7141 aaatggccca cttggcagta catcaatatc tattaatagt aacttggcaa gtacattact
     7201 attggaagta cgccagggta cattggcagt actcccattg acgtcaatgg cggtaaatgg
     7261 cccgcgatgg ctgccaagta catccccatt gacgtcaatg gggaggggca atgacgcaaa
     7321 tgggcgttcc attgacgtaa atgggcggta ggcgtgccta atgggaggtc tatataagca
     7381 atgctcgttt agggaac
//
