LOCUS       sequence-373757-        4862 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An wt EGFP control for Zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4862)
  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 4862)
  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-wt-EGFP
FEATURES             Location/Qualifiers
     source          1..4862
                     /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             join(85..87,88..90,91..801)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(85..87,88..90,91..801)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(130..151)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(391..410)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     738..759
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             811..840
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             856..873
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    914..1048
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(938..957)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     992..1011
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1161..1181)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1161..1179)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1200..1216)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1200..1216)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1213..1235)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1224..1240
                     /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(1248..1254,1255..1272,1273..1278))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1293..1314
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1431..1448)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1602..2190)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1682..1701)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2361..3152,3153..3221))
                     /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     2984..3003
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3222..3326)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3352..3807)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3489..3510)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3701..3720
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3878..4862
                     /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 agcaagggcg aggagctgtt caccggggtg
      121 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
      181 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
      241 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
      301 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
      361 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
      421 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
      481 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
      541 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
      601 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
      661 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
      721 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
      781 ggcatggacg agctgtacaa ggggcccttc gaacaaaaac tcatctcaga agaggatctg
      841 aatatgcata ccggtcatca tcaccatcac cattgactcg agcctctaga actatagtga
      901 gtcgtattac gtagatccag acatgataag atacattgat gagtttggac aaaccacaac
      961 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     1021 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     1081 ggttcagggg gaggtgtggg aggtttttta attcgcggcc gcggcgccaa tgcattgggc
     1141 ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     1201 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     1261 gggagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     1321 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     1381 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     1441 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     1501 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     1561 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     1621 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1681 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     1741 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     1801 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     1861 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     1921 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     1981 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     2041 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     2101 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     2161 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     2221 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     2281 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     2341 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     2401 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     2461 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     2521 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     2581 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     2641 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     2701 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     2761 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     2821 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     2881 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     2941 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     3001 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     3061 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     3121 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     3181 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     3241 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     3301 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     3361 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     3421 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     3481 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     3541 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     3601 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     3661 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     3721 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     3781 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     3841 aagggcgatc ggtgcgggcc tcttcgctat tacgccagtc gaccgccaat tcaatatggc
     3901 gtatatggac tcatgccaat tcaatatggt ggatctggac ctgtgccaat tcaatatggc
     3961 gtatatggac tcgtgccaat tcaatatggt ggatctggac cccagccaat tcaatatggc
     4021 ggacttggca ccatgccaat tcaatatggc ggacctggca ctgtgccaac tggggagggg
     4081 tctacttggc acggtgccaa gtttgaggag gggtcttggc cctgtgccaa gtccgccata
     4141 ttgaattggc atggtgccaa taatggcggc catattggct atatgccagg atcaatatat
     4201 aggcaatatc caatatggcc ctatgccaat atggctattg gccaggttca atactatgta
     4261 ttggccctat gccatatagt attccatata tgggttttcc tattgacgta gatagcccct
     4321 cccaatgggc ggtcccatat accatatatg gggcttccta ataccgccca tagccactcc
     4381 cccattgacg tcaatggtct ctatatatgg tctttcctat tgacgtcata tgggcggtcc
     4441 tattgacgta tatggcgcct cccccattga cgtcaattac ggtaaatggc ccgcctggct
     4501 caatgcccat tgacgtcaat aggaccaccc accattgacg tcaatgggat ggctcattgc
     4561 ccattcatat ccgttctcac gccccctatt gacgtcaatg acggtaaatg gcccacttgg
     4621 cagtacatca atatctatta atagtaactt ggcaagtaca ttactattgg aagtacgcca
     4681 gggtacattg gcagtactcc cattgacgtc aatggcggta aatggcccgc gatggctgcc
     4741 aagtacatcc ccattgacgt caatggggag gggcaatgac gcaaatgggc gttccattga
     4801 cgtaaatggg cggtaggcgt gcctaatggg aggtctatat aagcaatgct cgtttaggga
     4861 ac
//
