LOCUS       sequence-449334-        4860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of eGFP in zebrafish embryos..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4860)
  AUTHORS   Chen GR, Sive H, Bartel DP
  TITLE     A Seed Mismatch Enhances Argonaute2-Catalyzed Cleavage and Partially
            Rescues Severely Impaired Cleavage Found in Fish.
  JOURNAL   Mol Cell. 2017 Dec 21;68(6):1095-1107.e5. doi: 
            10.1016/j.molcel.2017.11.032.
  PUBMED    29272705
REFERENCE   2  (bases 1 to 4860)
  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"; doi: 
            "10.1016/j.molcel.2017.11"; journalName: "Mol Cell"; date: 
            "2017-12-21- 21"; volume: "68"; issue: "6"; pages: "1095-1107.e5"
COMMENT     Sequence Label: pCS2+-eGFP
FEATURES             Location/Qualifiers
     source          1..4860
                     /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"
     misc_feature    83..135
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      147..156
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(153..155,156..158,159..872)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(153..155,156..158,159..869)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(198..219)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(459..478)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     806..827
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    908..1042
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(932..951)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     986..1005
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1155..1175)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1155..1173)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1194..1210)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1194..1210)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1207..1229)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1218..1234
                     /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(1242..1248,1249..1266,1267..1272))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1287..1308
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1425..1442)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1596..2184)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1676..1695)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2355..3146,3147..3215))
                     /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     2978..2997
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3216..3320)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3346..3801)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3483..3504)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3695..3714
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3883..4860
                     /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 atctcgagct caagcttcga attctgcagt cgacggtacc
      121 gcgggcccgg gatccaccgg ccggtcgcca ccatggtgag caagggcgag gagctgttca
      181 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
      241 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
      301 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
      361 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
      421 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
      481 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
      541 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
      601 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
      661 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
      721 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
      781 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
      841 tcactctcgg catggacgag ctgtacaagt aaagcggccc tagaactata gtgagtcgta
      901 ttacgtagat ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat
      961 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     1021 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     1081 gggggaggtg tgggaggttt tttaattcgc ggccgcggcg ccaatgcatt gggcccggta
     1141 cccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     1201 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccgggagc
     1261 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1321 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1381 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     1441 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     1501 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     1561 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     1621 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     1681 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     1741 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     1801 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     1861 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     1921 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     1981 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     2041 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2101 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2161 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2221 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2281 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2341 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     2401 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     2461 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     2521 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     2581 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     2641 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     2701 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     2761 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     2821 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     2881 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     2941 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3001 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3061 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3121 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3181 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     3241 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3301 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     3361 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     3421 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     3481 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     3541 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     3601 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     3661 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     3721 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     3781 atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc
     3841 gatcggtgcg ggcctcttcg ctattacgcc agtcgatcga ccgccaattc aatatggcgt
     3901 atatggactc atgccaattc aatatggtgg atctggacct gtgccaattc aatatggcgt
     3961 atatggactc gtgccaattc aatatggtgg atctggaccc cagccaattc aatatggcgg
     4021 acttggcacc atgccaattc aatatggcgg acctggcact gtgccaactg gggaggggtc
     4081 tacttggcac ggtgccaagt ttgaggaggg gtcttggccc tgtgccaagt ccgccatatt
     4141 gaattggcat ggtgccaata atggcggcca tattggctat atgccaggat caatatatag
     4201 gcaatatcca atatggccct atgccaatat ggctattggc caggttcaat actatgtatt
     4261 ggccctatgc catatagtat tccatatatg ggttttccta ttgacgtaga tagcccctcc
     4321 caatgggcgg tcccatatac catatatggg gcttcctaat accgcccata gccactcccc
     4381 cattgacgtc aatggtctct atatatggtc tttcctattg acgtcatatg ggcggtccta
     4441 ttgacgtata tggcgcctcc cccattgacg tcaattacgg taaatggccc gcctggctca
     4501 atgcccattg acgtcaatag gaccacccac cattgacgtc aatgggatgg ctcattgccc
     4561 attcatatcc gttctcacgc cccctattga cgtcaatgac ggtaaatggc ccacttggca
     4621 gtacatcaat atctattaat agtaacttgg caagtacatt actattggaa gtacgccagg
     4681 gtacattggc agtactccca ttgacgtcaa tggcggtaaa tggcccgcga tggctgccaa
     4741 gtacatcccc attgacgtca atggggaggg gcaatgacgc aaatgggcgt tccattgacg
     4801 taaatgggcg gtaggcgtgc ctaatgggag gtctatataa gcaatgctcg tttagggaac
//
