LOCUS       sequence-343309-        4414 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for making mRNA for overexpressing eGFP (C-FLAG+HA) in frog
            oocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4414)
  AUTHORS   Xiang K, Bartel DP
  TITLE     The molecular basis of coupling between poly(A)-tail length and 
            translational efficiency.
  JOURNAL   Elife. 2021 Jul 2;10. pii: 66493. doi: 10.7554/eLife.66493.
  PUBMED    34213414
REFERENCE   2  (bases 1 to 4414)
  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: "Elife."; 
            date: "2021-07-2"; volume: "10. pii: 66493. doi"; pages: " 
            10.7554/eLife.66493"
COMMENT     Sequence Label: C255
FEATURES             Location/Qualifiers
     source          1..4414
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     5'UTR           16..58
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             join(70..72,73..75,76..786)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(70..72,73..75,76..786)
                     /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(115..136)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(376..395)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     723..744
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             787..810
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             811..837
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     3'UTR           840..965
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     promoter        complement(1285..1303)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1286..1303)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1321..1337)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1321..1337)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1334..1356)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1345..1361
                     /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(1369..1375,1376..1393,1394..1399))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1414..1435
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1552..1569)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1723..2311)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1803..1822)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2482..3273,3274..3342))
                     /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     3105..3124
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3343..3447)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3515..3533
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3571..3593)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3693..3712
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3779..4234)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3916..3937)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4128..4147
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4360..4382
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4374..4391
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4375..4391
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(4398..4414,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4398..4414,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaataca agcttcttgt tctttttgca gaagctcaga ataaacgctc aactttggca
       61 gatctgaaca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
      121 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
      181 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
      241 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
      301 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
      361 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
      421 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
      481 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
      541 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
      601 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
      661 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
      721 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
      781 tacaaggact acaaggacga tgacgataag tatccatatg atgttccaga ttatgcttaa
      841 accagcctca agaacacccg aatggagtct ctaagctaca taataccaac ttacacttta
      901 caaaatgttg tcccccaaaa tgtagccatt cgtatctgct cctaataaaa agaaagtttc
      961 ttcacattct gggcgtagat ccagacatga aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     1021 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     1081 aaaaaaaaac agtagggttg taaaggtttt tcttttcctg agaaaacaac cttttgtttt
     1141 ctcaggtttt gctttttggc ctttccctag ctttaaaaaa aaaagcaaaa gctagccatg
     1201 gtcccagcct cctcgctggc ggctagtggg caacatgctt cggcatggcg aatgggacgc
     1261 ggccgcatgc ataagcttga gtattctata gtgtcaccta aatagcttgg cgtaatcatg
     1321 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     1381 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     1441 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1501 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     1561 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     1621 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1681 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1741 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1801 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1861 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1921 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1981 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2041 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2101 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2161 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2221 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2281 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2341 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2401 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2461 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2521 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2581 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     2641 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2701 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2761 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     2821 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     2881 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     2941 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     3001 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     3061 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     3121 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     3181 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3241 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3301 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     3361 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3421 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     3481 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     3541 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     3601 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     3661 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     3721 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa
     3781 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     3841 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     3901 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     3961 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     4021 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     4081 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     4141 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     4201 cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact
     4261 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     4321 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     4381 cgacggccag tgaattgtaa tacgactcac tata
//
