LOCUS       sequence-56721-e        7463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7463)
  AUTHORS   Chu D, Kazana E, Bellanger N, Singh T, Tuite MF, von der Haar T
  TITLE     Translation elongation can control translation initiation on 
            eukaryotic mRNAs.
  JOURNAL   EMBO J. 2014 Jan 1;33(1):21-34. doi: 10.1002/embj.201385651. Epub 
            2013 Dec 19.
  PUBMED    24357599
REFERENCE   2  (bases 1 to 7463)
  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: 
            "10.1002/embj.201385651"; journalName: "EMBO J"; date: "2014-01-1- 
            1"; volume: "33"; issue: "1"; pages: "21-34"
COMMENT     Sequence Label: pTH646-2µ-RLuc
FEATURES             Location/Qualifiers
     source          1..7463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..29)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(183..771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(263..282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(942..1733,1734..1802))
                     /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     1565..1584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1803..1907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1975..1993
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2018..2238
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             2239..3042
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      3153..4499
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(4524..4823)
                     /label=ADH2 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 2 (ADH2) gene"
     CDS             complement(4854..5786)
                     /codon_start=1
                     /product="luciferase from the anthozoan coelenterate 
                     Renilla reniformis (sea pansy)"
                     /label=Rluc
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     promoter        6198..6841
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     6734..6757
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     terminator      7010..7197
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(7065..7089)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(7244..7260)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7244..7260)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7257..7279)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7268..7284
                     /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(7292..7298,7299..7316,7317..7322))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7337..7358
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
       61 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      601 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
      661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1081 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1801 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1861 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1921 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1981 cgtatcacga ggccctttcg tcttcaagaa ttagcttttc aattcaattc atcatttttt
     2041 ttttattctt ttttttgatt tcggtttctt tgaaattttt ttgattcggt aatctccgaa
     2101 cagaaggaag aacgaaggaa ggagcacaga cttagattgg tatatatacg catatgtagt
     2161 gttgaagaaa catgaaattg cccagtattc ttaacccaac tgcacagaac aaaaacatgc
     2221 aggaaacgaa gataaatcat gtcgaaagct acatataagg aacgtgctgc tactcatcct
     2281 agtcctgttg ctgccaagct atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct
     2341 tcattggatg ttcgtaccac caaggaatta ctggagttag ttgaagcatt aggtcccaaa
     2401 atttgtttac taaaaacaca tgtggatatc ttgactgatt tttccatgga gggcacagtt
     2461 aagccgctaa aggcattatc cgccaagtac aattttttac tcttcgaaga cagaaaattt
     2521 gctgacattg gtaatacagt caaattgcag tactctgcgg gtgtatacag aatagcagaa
     2581 tgggcagaca ttacgaatgc acacggtgtg gtgggcccag gtattgttag cggtttgaag
     2641 caggcggcag aagaagtaac aaaggaacct agaggccttt tgatgttagc agaattgtca
     2701 tgcaagggct ccctatctac tggagaatat actaagggta ctgttgacat tgcgaagagc
     2761 gacaaagatt ttgttatcgg ctttattgct caaagagaca tgggtggaag agatgaaggt
     2821 tacgattggt tgattatgac acccggtgtg ggtttagatg acaagggaga cgcattgggt
     2881 caacagtata gaaccgtgga tgatgtggtc tctacaggat ctgacattat tattgttgga
     2941 agaggactat ttgcaaaggg aagggatgct aaggtagagg gtgaacgtta cagaaaagca
     3001 ggctgggaag catatttgag aagatgcggc cagcaaaact aaaaaactgt attataagta
     3061 aatgcatgta tactaaactc acaaattaga gcttcaattt aattatatca gttattaccc
     3121 aattctcatg tttgacagct tatcatcgga tcgatccaat atcaaaggaa atgatagcat
     3181 tgaaggatga gactaatcca attgaggagt ggcagcatat agaacagcta aagggtagtg
     3241 ctgaaggaag catacgatac cccgcatgga atgggataat atcacaggag gtactagact
     3301 acctttcatc ctacataaat agacgcatat aagtacgcat ttaagcataa acacgcacta
     3361 tgccgttctt ctcatgtata tatatataca ggcaacacgc agatataggt gcgacgtgaa
     3421 cagtgagctg tatgtgcgca gctcgcgttg cattttcgga agcgctcgtt ttcggaaacg
     3481 ctttgaagtt cctattccga agttcctatt ctctagctag aaagtatagg aacttcagag
     3541 cgcttttgaa aaccaaaagc gctctgaaga cgcactttca aaaaaccaaa aacgcaccgg
     3601 actgtaacga gctactaaaa tattgcgaat accgcttcca caaacattgc tcaaaagtat
     3661 ctctttgcta tatatctctg tgctatatcc ctatataacc tacccatcca cctttcgctc
     3721 cttgaacttg catctaaact cgacctctac attttttatg tttatctcta gtattactct
     3781 ttagacaaaa aaattgtagt aagaactatt catagagtga atcgaaaaca atacgaaaat
     3841 gtaaacattt cctatacgta gtatatagag acaaaataga agaaaccgtt cataattttc
     3901 tgaccaatga agaatcatca acgctatcac tttctgttca caaagtatgc gcaatccaca
     3961 tcggtataga atataatcgg ggatgccttt atcttgaaaa aatgcacccg cagcttcgct
     4021 agtaatcagt aaacgcggga agtggagtca ggcttttttt atggaagaga aaatagacac
     4081 caaagtagcc ttcttctaac cttaacggac ctacagtgca aaaagttatc aagagactgc
     4141 attatagagc gcacaaagga gaaaaaaagt aatctaagat gctttgttag aaaaatagcg
     4201 ctctcgggat gcatttttgt agaacaaaaa agaagtatag attctttgtt ggtaaaatag
     4261 cgctctcgcg ttgcatttct gttctgtaaa aatgcagctc agattctttg tttgaaaaat
     4321 tagcgctctc gcgttgcatt tttgttttac aaaaatgaag cacagattct tcgttggtaa
     4381 aatagcgctt tcgcgttgca tttctgttct gtaaaaatgc agctcagatt ctttgtttga
     4441 aaaattagcg ctctcgcgtt gcatttttgt tctacaaaat gaagcacaga tgcttcgttc
     4501 cgctcgaggg catgcgaagg aaaatgagaa atatcgaggg agacgattca gaggagcagg
     4561 acaaactata accgactgtt tgttggagga tgccgtacat aacgaacact gctgaagcta
     4621 ccatgtctac agtttagagg aatgggtaca actcacaggc gagggatggt gttcactcgt
     4681 gctagcaaac gcggtgggag caaaaagtag aatattatct tttattcgtg aaacttcgaa
     4741 cactgtcatc taaagatgct atatactaat ataggcatac ttgataatgg aaactataaa
     4801 tcgtgaaggc ataggagatc cgcttattta gaagtgtcga attcttacga catttgttca
     4861 tttttgagaa ctcgctcaac gaacgatttg atatattttc ccatttcatc aggtgcatct
     4921 tcttgcgaaa aatgaagacc ttttactttg acaaattcag tattaggaaa cttcttggca
     4981 ccttcaacaa tagcattgga aaagaatcct gggtccgatt caataaacat ttttggtaaa
     5041 tcatcacttg cacgtagata agcattataa ttcctaacaa tttgtacaac gtcaggttta
     5101 ccacctttta ctaacgggat ttcacgaggc catgataatg ttggacgacg aacttcacct
     5161 ttctctttga atggttcaag atatgctgca aattcttctg gttctaactt tctcatgatt
     5221 tttgatggca acatggtttc cacgaagaag ttattctcca aaaccatttt ttctccttct
     5281 tcagatttga tcaacgcaat atcttcttca atatcaggcc attcatccca tgattcaatc
     5341 acatctacta cactttcagc gtgaactatt gctttgatct tatcttgatg ctcatagcta
     5401 taatgaaatg ccaaacaagc accccaatca tggccgacaa aaatgatctt ctttggtaaa
     5461 ttaagaagtt caaaccatgc agtaagatat ttgtaatgat caagtaacct ataagaacca
     5521 ttaccagatt tgcctgattt gcccatacca ataaggtctg gtataataca ccgcgctact
     5581 ggctcaatat gtggcacaac atgtcgccat aaataagaag aggccgcgtt accatgtaaa
     5641 aaaataacag cattttctgc atgtttttct gaatcataat aattaataaa tgaatcaaga
     5701 acattcattt gtttacatct ggcccaccac tgcggaccag ttatcatccg tttcctttgt
     5761 tctggatcat aaactttcga agtcatcccg gggagttgat tgtagcttgg tatagcttga
     5821 aatattgtgc agaaaaagaa acaaggaaga aagggaacga gaacaatgac gaggaaacaa
     5881 aagattaata attgcaggtc tatttatact tgatagcaag acagcaaact ttttttattt
     5941 caaattcaag taactggaag gaaggccgta taccgttgct cattagagag tagtgtgcgt
     6001 gaatgaagga aggaaaaagt ttcgtgtgtt cgaagatacc cctcatcagc tctggaacaa
     6061 cgacatctgt tggtgctgtc tttgtcgtta attttttcct ttagtgtctt ccatcatttt
     6121 ttttgtcatt gcggatatgg tgagacaaca acgggggaga gagaaaagaa aaaaaaagaa
     6181 aagaatccgg gagtttatca ttatcaatac tgccatttca aagaatacgt aaataattaa
     6241 tagtagtgat tttcctaact ttatttagtc aaaaaattag ccttttaatt ctgctgtaac
     6301 ccgtacatgc ccaaaatagg gggcgggtta cacagaatat ataacatcgt aggtgtctgg
     6361 gtgaacagtt tattcctggc atccactaaa tataatggag cccgcttttt aagctggcat
     6421 ccagaaaaaa aaagaatccc agcaccaaaa tattgttttc ttcaccaacc atcagttcat
     6481 aggtccattc tcttagcgca actacagaga acaggggcac aaacaggcaa aaaacgggca
     6541 caacctcaat ggagtgatgc aacctgcctg gagtaaatga tgacacaagg caattgaccc
     6601 acgcatgtat ctatctcatt ttcttacacc ttctattacc ttctgctctc tctgatttgg
     6661 aaaaagctga aaaaaaaggt tgaaaccagt tccctgaaat tattccccta cttgactaat
     6721 aagtatataa agacggtagg tattgattgt aattctgtaa atctatttct taaacttctt
     6781 aaattctact tttatagtta gtcttttttt tagttttaaa acaccaagaa cttagtttcg
     6841 acggatcctc tagagtcgac ctgcaggcat gcaagctttg gacttcttcg ccagaggttt
     6901 ggtcaagtct ccaatcaagg ttgtcggctt gtctaccttg ccagaaattt acgaaaagat
     6961 ggaaaagggt caaatcgttg gtagatacgt tgttgacact tctaaataag cgaatttctt
     7021 atgatttatg atttttatta ttaaataagt tataaaaaaa ataagtgtat acaaatttta
     7081 aagtgactct taggttttaa aacgaaaatt cttattcttg agtaactctt tcctgtaggt
     7141 caggttgctt tctcaggtat agcatgaggt cgctcttatt gaccacacct ctaccggcat
     7201 gccggaaatt cccctaccct atgaaaagct tggcgtaatc atggtcatag ctgtttcctg
     7261 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     7321 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     7381 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     7441 gaggcggttt gcgtattggg cgc
//
