LOCUS       sequence-75545-e        4016 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template DNA for in vitro transcription using T7 polymerase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4016)
  AUTHORS   Fukaya T, Tomari Y
  TITLE     PABP is not essential for microRNA-mediated translational repression
            and deadenylation in vitro.
  JOURNAL   EMBO J. 2011 Nov 25;30(24):4998-5009. doi: 10.1038/emboj.2011.426.
  PUBMED    22117217
REFERENCE   2  (bases 1 to 4016)
  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: 
            "10.1038/emboj.2011"; journalName: "EMBO J"; date: "2011-11-25- 25";
            volume: "30"; issue: "24"; pages: "4998-5009"
COMMENT     Sequence Label: pUC57-Rluc-BoxB-A114-N40
FEATURES             Location/Qualifiers
     source          1..4016
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        408..426
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      430..439
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             436..1371
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     protein_bind    1425..1443
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    1467..1485
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    1491..1509
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    1549..1567
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    1573..1591
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     primer_bind     complement(1795..1811)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1795..1811)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1808..1830)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1819..1835
                     /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(1843..1849,1850..1867,1868..1873))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1888..1909
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2026..2043)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2197..2785)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2277..2296)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2956..3747,3748..3816))
                     /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     3579..3598
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3817..3921)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3989..4007
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctctaa tacgactcac
      421 tataggctag ccaccatggc ttccaaggtg tacgaccccg agcaacgcaa acgcatgatc
      481 actgggcctc agtggtgggc tcgctgcaag caaatgaacg tgctggactc cttcatcaac
      541 tactatgatt ccgagaagca cgccgagaac gccgtgattt ttctgcatgg taacgctgcc
      601 tccagctacc tgtggaggca cgtcgtgcct cacatcgagc ccgtggctag atgcatcatc
      661 cctgatctga tcggaatggg taagtccggc aagagcggga atggctcata tcgcctcctg
      721 gatcactaca agtacctcac cgcttggttc gagctgctga accttccaaa gaaaatcatc
      781 tttgtgggcc acgactgggg ggcttgtctg gcctttcact actcctacga gcaccaagac
      841 aagatcaagg ccatcgtcca tgctgagagt gtcgtggacg tgatcgagtc ctgggacgag
      901 tggcctgaca tcgaggagga tatcgccctg atcaagagcg aagagggcga gaaaatggtg
      961 cttgagaata acttcttcgt cgagaccatg ctcccaagca agatcatgcg gaaactggag
     1021 cctgaggagt tcgctgccta cctggagcca ttcaaggaga agggcgaggt tagacggcct
     1081 accctctcct ggcctcgcga gatccctctc gttaagggag gcaagcccga cgtcgtccag
     1141 attgtccgca actacaacgc ctaccttcgg gccagcgacg atctgcctaa gatgttcatc
     1201 gagtccgacc ctgggttctt ttccaacgct attgtcgagg gagctaagaa gttccctaac
     1261 accgagttcg tgaaggtgaa gggcctccac ttcagccagg aggacgctcc agatgaaatg
     1321 ggtaagtaca tcaagagctt cgtggagcgc gtgctgaaga acgagcagta attctaggcg
     1381 atcgctcgag ttccctaagt ccaactacca aactggggat tcctgggccc tgaagaaggg
     1441 cccctcgact agtccaaata caaactgggc cctgaagaag ggcccatata gggccctgaa
     1501 gaagggccct atcgaggata ttatctcgac taagtccaac tacaaactgg gccctgaaga
     1561 agggcccata tagggccctg aagaagggcc ctatcgagga tattatgcgg ccgctggccg
     1621 cctgcagaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     1681 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     1741 atgcatctgg tttcctggtc tgtctcctct cggtttaagc ttggcgtaat catggtcata
     1801 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     1861 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     1921 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1981 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     2041 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2101 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2161 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2221 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2281 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2341 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2401 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2461 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2521 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2581 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     2641 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2701 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2761 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2821 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2881 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2941 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3001 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3061 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3121 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3181 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3241 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3301 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3361 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3421 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3481 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3541 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3601 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3661 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3721 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3781 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     3841 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3901 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     3961 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
