LOCUS       sequence-460233-        7071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent reporter to measure ISR state-dependent protein 
            synthesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7071)
  AUTHORS   Oliver ML, Caffall ZF, Eatman CB, Faw TD, Calakos N
  TITLE     DIO-SPOTlight Transgenic Mouse to Functionally Monitor Protein 
            Synthesis Regulated by the Integrated Stress Response
  JOURNAL   eLife 2025 14:RP104457
REFERENCE   2  (bases 1 to 7071)
  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 2025 
            14:RP104457"
COMMENT     Sequence Label: AAV-DIO-SPOTlight-U2GCR
FEATURES             Location/Qualifiers
     source          1..7071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     protein_bind    137..157
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        158..443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          718..945
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     protein_bind    950..974
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(975..1008)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1041..1074)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1173..1196)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     1640..1658
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(2518..2547)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(2651..2707)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(join(2726..3436,3437..3439,3440..3442,
                     3443..3445))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     CDS             complement(join(2726..3436,3437..3439,3440..3442,
                     3443..3445))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     primer_bind     3117..3136
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3376..3397
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3541..3550
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3954..3987
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    4020..4053
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(4054..4078)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(4102..4119)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4108..4315
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4323..4463
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4323..4452
                     /label=AAV2 ITR
     rep_origin      4538..4993
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4625..4644)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4835..4856
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5010..5029)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5129..5151
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5189..5207)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5275..5379
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5380..5448,5449..6240)
                     /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     complement(5598..5617)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6411..6999
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6900..6919
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(7061..7071,1..130)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tctagacaac tttgtataga aaagttgcgt tacataactt acggtaaatg
      181 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag
      241 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      301 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      361 cctggcattg tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      421 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      481 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      541 cgatgggggc gggggggggg ggggggcgcg ccccaggggg gggggggggg gggggggggg
      601 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      661 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      721 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      781 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      841 ggctgtaatt agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta
      901 atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg caagtttgta
      961 caaaaaagca ggctataact tcgtatagga tactttatac gaagttatcc tcgagtaccg
     1021 gtcacgcgta cgcaagcttg ataacttcgt atagcataca ttatacgaag ttatcctagg
     1081 gccaccttac ttgtacagct cgtccatgcc gtacaggaac aggtggtggc ggccctcgga
     1141 gcgctcgtac tgttccacga tggtgtagtc ctcgttgtgg gaggtgatgt ccagcttggt
     1201 gtccacgtag tagtagccgg gcagttgcac gggcttcttg gccatgtaga tggtcttgaa
     1261 ctccaccagg tagcggccgc cgtccttcag cttcagggcc tggtggatct cgcccttcag
     1321 cacgccgtcg cgggggtaca ggcgctcggt ggaggcctcc cagcccatgg tcttcttctg
     1381 cattacgggg ccgtcggggg ggaagtttgt gccgcgcatc ttcaccttgt agatcagcgt
     1441 gccgtcctgc agggaggagt cctgggtcac ggtcaccaga ccgccgtcct cgaagttcat
     1501 cacgcgctcc cacttgaagc cctcggggaa ggacagcttc ttgtaatcgg ggatgtcggc
     1561 ggggtgcttc acgtacgcct tggagccgta catgaactgg ggggacagga tgtcccaggc
     1621 gaagggcagg gggccgccct tggtcacctt cagcttggcg gtctgggtgc cctcgtaggg
     1681 gcggccctcg ccctcgccct cgatctcgaa ctcgtggccg ttcatggagc cctccatgcg
     1741 caccttgaag cgcatgaact ctttgatgac ggccatgttg ttgtcctcgg aggaggcggt
     1801 gccggagctg ccactaccag tactaccagt accgtgtcct aggaacaagt gatggcgtcc
     1861 ctctgatcgc tcgtattgtt caacgatagt gtagtcttca ttatgggatg ttatgtccaa
     1921 cttagtgtcg acgtaatagt aaccaggcaa ctgcacaggc ttcttagcca tatagatagt
     1981 tttaaattca acaaggtaat ggccgccgtc cttgagttta agagcctggt ggatctcgcc
     2041 tttgaggaca ccatccctag gataaagtcg ctcagtgctg gcctcccagc ccattgtctt
     2101 cttttgcata acgggaccgt cgggggggaa gttggtacct ctcatcttaa ctttgtaaat
     2161 caatgtaccg tcttgaaggc tagagtcttg ggttactgtt acgagccccc cgtcttcaaa
     2221 attcatcact cgctcccatt tgaacccttc tgggaaggaa agtttcttat aatctggaat
     2281 atcggcggga tgtttgacat atgccttact gccgtacata aactgaggac tgaggatgtc
     2341 ccatgcaaat ggcaaaggtc cgccttttgt gactttcaat ttagcggtct gagtgccctc
     2401 gtaaggtcgt ccctctccct ccccttcgat ttcaaattcg tggccgttca tagatccctc
     2461 cattcgtacc ttgaaccgca taaactcctt aatcacctcc tcgcccttgc taaccaccag
     2521 atcctcttct gagatgagtt tttgttcagt catgttgtgg ggctttgctg gattcgagga
     2581 atgtgcttaa ctcgaaggta tctttgtccg ttacagcaac actgctgctg gatttcgtga
     2641 agagcgccac aggtccaggg ttctcctcca cgtctccagc ctgcttcagc aggctgaagt
     2701 tagtagctcc gcttcctcca ctacccttgt acagctcgtc catgccgaga gtgatcccgg
     2761 cggcggtcac gaactccagc aggaccatat gatcgcgctt ctcgttgggg tctttgctca
     2821 gggcggactg ggtgctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg
     2881 gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga
     2941 tcttgaagtt caccttgatg ccgttcttct gcttgtcggc cattatatag acgttgtggc
     3001 tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc
     3061 ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct
     3121 tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatcg
     3181 cactcttgaa gaagtcgtgc tgcttcatat gatcggggta gcggctgaag cactgcacgc
     3241 cgtaggtcag ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga
     3301 tgaacttcag ggtcagcttg ccgtaggtgg cgtcgccctc gccctcgccg gacacgctga
     3361 acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca
     3421 gctcctcgcc cttgctcacc gccatggctt aggccggtgg gggttgccgc tgcagagcct
     3481 ggtgctgctg ccgccgctgc aaaggccaac gctgccgcag gcactgctgc ctctaatacg
     3541 ccatggtggc cgcaagccct gcgggagggg aagaggaaag ggacacccga gcacacaact
     3601 taaaccggca gacagcaagc agaaatggcg gcggcacgcc ctaaacccgc cctttatagc
     3661 ctcggccagg gacgcagcac accgcgtatc ctcccactgt ggtcacgtga tcctaccgca
     3721 gcgccaccaa accgcctctc actgacgccc tcgcccgccc caacccttct ccgctgtgct
     3781 cggggggtaa ctgtggcgtt agagatcgtc ctaaaggccc catgagagaa aagtgcacta
     3841 ctttataggt tgacgaccag ccgcggtctg gttccggagc gcggagggat cgctcgttga
     3901 cgaatttccg gtgtcttaca aggcggggtg ggacacctag ccggatctct agaataactt
     3961 cgtataaagt atcctatacg aagttatagg tacctgagct caacatcgat agcactagta
     4021 taacttcgta taatgtatgc tatacgaagt tatacccagc tttcttgtac aaagtgggaa
     4081 ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     4141 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     4201 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     4261 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggagggcc
     4321 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     4381 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     4441 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     4501 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     4561 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     4621 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4681 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4741 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4801 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4861 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     4921 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     4981 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5041 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5101 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5161 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     5221 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5281 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5341 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5401 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5461 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5521 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5581 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5641 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5701 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5761 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5821 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5881 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5941 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6001 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6061 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     6121 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6181 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6241 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6301 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6361 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6421 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6481 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6541 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6601 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6661 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6721 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6781 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6841 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6901 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6961 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7021 ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca g
//
