LOCUS       sequence-450177-        7454 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoterless MET predicted IRES between Rluc and Fluc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7454)
  AUTHORS   Cerqua M, Foiani M, Boccaccio C, Comoglio PM, Altintas DM
  TITLE     The integrated stress response drives MET oncogene overexpression in
            cancers.
  JOURNAL   EMBO J. 2025 Jan 7. doi: 10.1038/s44318-024-00338-4.
  PUBMED    39774381
REFERENCE   2  (bases 1 to 7454)
  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: "EMBO J. 
            2025 Jan 7. doi: 10.1038/s44318-024-00338-4."
COMMENT     Sequence Label: RLuc_MET_95_FLuc_Pless
FEATURES             Location/Qualifiers
     source          1..7454
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             10..945
                     /codon_start=1
                     /product="luciferase from the anthozoan coelenterate 
                     Renilla reniformis (sea pansy)"
                     /label=Rluc
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             1041..2693
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     primer_bind     complement(1107..1125)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     promoter        complement(2776..2794)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2777..2794)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2814..2831)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2820..3044
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3090..3518
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3177..3196)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3387..3408
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3527..3547)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3532..3861
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3712..3847
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3774..3793
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3928..4722
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3982..4001)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4592..4611
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4896..5017
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4933..4952)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4987..5006
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5066..5082)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5066..5082)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5079..5101)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5090..5106
                     /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(5114..5120,5121..5138,5139..5144))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5159..5180
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5297..5314)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5468..6056)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5548..5567)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6227..7018,7019..7087))
                     /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     6850..6869
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7088..7192)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7267..7286)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctagccacca tgacttcgaa agtttatgat ccagaacaaa ggaaacggat gataactggt
       61 ccgcagtggt gggccagatg taaacaaatg aatgttcttg attcatttat taattattat
      121 gattcagaaa aacatgcaga aaatgctgtt atttttttac atggtaacgc ggcctcttct
      181 tatttatggc gacatgttgt gccacatatt gagccagtag cgcggtgtat tataccagac
      241 cttattggta tgggcaaatc aggcaaatct ggtaatggtt cttataggtt acttgatcat
      301 tacaaatatc ttactgcatg gtttgaactt cttaatttac caaagaagat catttttgtc
      361 ggccatgatt ggggtgcttg tttggcattt cattatagct atgagcatca agataagatc
      421 aaagcaatag ttcacgctga aagtgtagta gatgtgattg aatcatggga tgaatggcct
      481 gatattgaag aagatattgc gttgatcaaa tctgaagaag gagaaaaaat ggttttggag
      541 aataacttct tcgtggaaac catgttgcca tcaaaaatca tgagaaagtt agaaccagaa
      601 gaatttgcag catatcttga accattcaaa gagaaaggtg aagttcgtcg tccaacatta
      661 tcatggcctc gtgaaatccc gttagtaaaa ggtggtaaac ctgacgttgt acaaattgtt
      721 aggaattata atgcttatct acgtgcaagt gatgatttac caaaaatgtt tattgaatcg
      781 gacccaggat tcttttccaa tgctattgtt gaaggtgcca agaagtttcc taatactgaa
      841 tttgtcaaag taaaaggtct tcatttttcg caagaagatg cacctgatga aatgggaaaa
      901 tatatcaaat cgttcgttga gcgagttctc aaaaatgaac aataactgag aggaggcggg
      961 gaggcgcgga gcgcgcgtgt ggtccttgcg ccgctgactt ctccactggt tcctgggcac
     1021 cgaaagataa acctctcata atggaagacg ccaaaaacat aaagaaaggc ccggcgccat
     1081 tctatcctct agaggatgga accgctggag agcaactgca taaggctatg aagagatacg
     1141 ccctggttcc tggaacaatt gcttttacag atgcacatat cgaggtgaac atcacgtacg
     1201 cggaatactt cgaaatgtcc gttcggttgg cagaagctat gaaacgatat gggctgaata
     1261 caaatcacag aatcgtcgta tgcagtgaaa actctcttca attctttatg ccggtgttgg
     1321 gcgcgttatt tatcggagtt gcagttgcgc ccgcgaacga catttataat gaacgtgaat
     1381 tgctcaacag tatgaacatt tcgcagccta ccgtagtgtt tgtttccaaa aaggggttgc
     1441 aaaaaatttt gaacgtgcaa aaaaaattac caataatcca gaaaattatt atcatggatt
     1501 ctaaaacgga ttaccaggga tttcagtcga tgtacacgtt cgtcacatct catctacctc
     1561 ccggttttaa tgaatacgat tttgtaccag agtcctttga tcgtgacaaa acaattgcac
     1621 tgataatgaa ttcctctgga tctactgggt tacctaaggg tgtggccctt ccgcatagaa
     1681 ctgcctgcgt cagattctcg catgccagag atcctatttt tggcaatcaa atcattccgg
     1741 atactgcgat tttaagtgtt gttccattcc atcacggttt tggaatgttt actacactcg
     1801 gatatttgat atgtggattt cgagtcgtct taatgtatag atttgaagaa gagctgtttt
     1861 tacgatccct tcaggattac aaaattcaaa gtgcgttgct agtaccaacc ctattttcat
     1921 tcttcgccaa aagcactctg attgacaaat acgatttatc taatttacac gaaattgctt
     1981 ctgggggcgc acctctttcg aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc
     2041 cagggatacg acaaggatat gggctcactg agactacatc agctattctg attacacccg
     2101 agggggatga taaaccgggc gcggtcggta aagttgttcc attttttgaa gcgaaggttg
     2161 tggatctgga taccgggaaa acgctgggcg ttaatcagag aggcgaatta tgtgtcagag
     2221 gacctatgat tatgtccggt tatgtaaaca atccggaagc gaccaacgcc ttgattgaca
     2281 aggatggatg gctacattct ggagacatag cttactggga cgaagacgaa cacttcttca
     2341 tagttgaccg cttgaagtct ttaattaaat acaaaggata tcaggtggcc cccgctgaat
     2401 tggaatcgat attgttacaa caccccaaca tcttcgacgc gggcgtggca ggtcttcccg
     2461 acgatgacgc cggtgaactt cccgccgccg ttgttgtttt ggagcacgga aagacgatga
     2521 cggaaaaaga gatcgtggat tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg
     2581 gaggagttgt gtttgtggac gaagtaccga aaggtcttac cggaaaactc gacgcaagaa
     2641 aaatcagaga gatcctcata aaggccaaga agggcggaaa gtccaaattg taaaatgtaa
     2701 ctgtattcag cgatgacgaa attcttagct attgtaatcc tccgaggcct cgagcatgca
     2761 tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac
     2821 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2881 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2941 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3001 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3061 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3121 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3181 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3241 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3301 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3361 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3421 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3481 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3541 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3601 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3661 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3721 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3781 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     3841 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     3901 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     3961 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4021 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4081 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4141 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4201 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4261 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4321 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4381 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4441 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4501 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4561 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4621 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4681 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     4741 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     4801 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4861 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     4921 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4981 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5041 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5101 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5161 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5221 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5281 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5341 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5401 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5461 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5521 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5581 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5641 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5701 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5761 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5821 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5881 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5941 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6001 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6061 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6121 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6181 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6241 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6301 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6361 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6421 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6481 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6541 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6601 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6661 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6721 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6781 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6841 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6901 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6961 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7021 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7081 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7141 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7201 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
     7261 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     7321 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     7381 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     7441 ggccagatat acgc
//
