LOCUS       sequence-334277-        6889 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter to measure ISR state-dependent protein synthesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6889)
  AUTHORS   Helseth AR, Hernandez-Martinez R, Hall VL, Oliver ML, Turner BD, 
            Caffall ZF, Rittiner JE, Shipman MK, King CS, Gradinaru V, Gerfen C,
            Costa-Mattioli M, Calakos N
  TITLE     Cholinergic neurons constitutively engage the ISR for dopamine 
            modulation and skill learning in mice.
  JOURNAL   Science. 2021 Apr 23;372(6540). pii: 372/6540/eabe1931. doi: 
            10.1126/science.abe1931.
  PUBMED    33888613
REFERENCE   2  (bases 1 to 6889)
  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: "Science."; 
            date: "2021-04-23"; volume: "372(6540). pii: 372/6540/eabe1931. 
            doi"; pages: " 10.1126/science.abe1931"
COMMENT     Sequence Label: AAV-CAG-SPOTlight-U2GCR
FEATURES             Location/Qualifiers
     source          1..6889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        441..718
                     /label=chicken beta-actin promoter
     primer_bind     997..1019
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1081..1100
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1525..1534
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1630..1632,1633..1635,1636..1638,1639..2349)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     CDS             join(1630..1632,1633..1635,1636..1638,1639..2349)
                     /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     complement(1678..1699)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1939..1958)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2368..2424
                     /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             2528..2557
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3417..3435)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3879..3902
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     repeat_region   4247..4376
                     /label=AAV2 ITR
     rep_origin      4448..4903
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4535..4554)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4745..4766
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4929..5033
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5034..5102,5103..5894)
                     /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(5252..5271)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6065..6653
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6554..6573
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 aggaagagta ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      421 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc
      481 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat
      541 gggggcgggg gggggggggg ggcgcgcgcc ggggggggsg gggsggggsg rggggsgggg
      601 sggggsgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc
      661 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg
      721 agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
      781 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      841 gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag
      901 ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggc tgtccgcggg
      961 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     1021 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
     1081 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattggat ccggctaggt
     1141 gtcccacccc gccttgtaag acaccggaaa ttcgtcaacg agcgatccct ccgcgctccg
     1201 gaaccagacc gcggctggtc gtcaacctat aaagtagtgc acttttctct catggggcct
     1261 ttaggacgat ctctaacgcc acagttaccc cccgagcaca gcggagaagg gttggggcgg
     1321 gcgagggcgt cagtgagagg cggtttggtg gcgctgcggt aggatcacgt gaccacagtg
     1381 ggaggatacg cggtgtgctg cgtccctggc cgaggctata aagggcgggt ttagggcgtg
     1441 ccgccgccat ttctgcttgc tgtctgccgg tttaagttgt gtgctcgggt gtccctttcc
     1501 tcttcccctc ccgcagggct tgcggccacc atggcgtatt agaggcagca gtgcctgcgg
     1561 cagcgttggc ctttgcagcg gcggcagcag caccaggctc tgcagcggca acccccaccg
     1621 gcctaagcca tggcggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1681 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1741 gacgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1801 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     1861 gatcatatga agcagcacga cttcttcaag agtgcgatgc ccgaaggcta cgtccaggag
     1921 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1981 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     2041 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat aatggccgac
     2101 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     2161 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     2221 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     2281 gatcatatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     2341 ctgtacaagg gtagtggagg aagcggagct actaacttca gcctgctgaa gcaggctgga
     2401 gacgtggagg agaaccctgg acctgtggcg ctcttcacga aatccagcag cagtgttgct
     2461 gtaacggaca aagatacctt cgagttaagc acattcctcg aatccagcaa agccccacaa
     2521 catgactgaa caaaaactca tctcagaaga ggatctggtg gttagcaagg gcgaggaggt
     2581 gattaaggag tttatgcggt tcaaggtacg aatggaggga tctatgaacg gccacgaatt
     2641 tgaaatcgaa ggggagggag agggacgacc ttacgagggc actcagaccg ctaaattgaa
     2701 agtcacaaaa ggcggacctt tgccatttgc atgggacatc ctcagtcctc agtttatgta
     2761 cggcagtaag gcatatgtca aacatcccgc cgatattcca gattataaga aactttcctt
     2821 cccagaaggg ttcaaatggg agcgagtgat gaattttgaa gacggggggc tcgtaacagt
     2881 aacccaagac tctagccttc aagacggtac attgatttac aaagttaaga tgagaggtac
     2941 caacttcccc cccgacggtc ccgttatgca aaagaagaca atgggctggg aggccagcac
     3001 tgagcgactt tatcctaggg atggtgtcct caaaggcgag atccaccagg ctcttaaact
     3061 caaggacggc ggccattacc ttgttgaatt taaaactatc tatatggcta agaagcctgt
     3121 gcagttgcct ggttactatt acgtcgacac taagttggac ataacatccc ataatgaaga
     3181 ctacactatc gttgaacaat acgagcgatc agagggacgc catcacttgt tcctaggaca
     3241 cggtactggt agtactggta gtggcagctc cggcaccgcc tcctccgagg acaacaacat
     3301 ggccgtcatc aaagagttca tgcgcttcaa ggtgcgcatg gagggctcca tgaacggcca
     3361 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     3421 gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt ccccccagtt
     3481 catgtacggc tccaaggcgt acgtgaagca ccccgccgac atccccgatt acaagaagct
     3541 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggtctggt
     3601 gaccgtgacc caggactcct ccctgcagga cggcacgctg atctacaagg tgaagatgcg
     3661 cggcacaaac ttcccccccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     3721 ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc accaggccct
     3781 gaagctgaag gacggcggcc gctacctggt ggagttcaag accatctaca tggccaagaa
     3841 gcccgtgcaa ctgcccggct actactacgt ggacaccaag ctggacatca cctcccacaa
     3901 cgaggactac accatcgtgg aacagtacga gcgctccgag ggccgccacc acctgttcct
     3961 gtacggcatg gacgagctgt acaagtaata gggtagtcag gtgctttgtg cttgtacata
     4021 gtcttgtgtt gctgtgtttg ctgtaataaa ttattttgta gtgaaagtac ctatgtgggt
     4081 tttcagtgtc attcaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg
     4141 ttttttaaag caagtaaaac ctctacaaat gtggtaaaat cgataaggat cttcctagag
     4201 catggctacg tagataagta gcatggcggg ttaatcatta actacaagga acccctagtg
     4261 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4321 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgg
     4381 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     4441 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4501 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4561 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4621 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     4681 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4741 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     4801 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     4861 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     4921 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4981 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5041 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5101 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5161 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5221 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5281 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5341 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5401 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5461 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5521 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5581 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5641 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     5701 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     5761 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5821 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     5881 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     5941 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6001 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6061 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6121 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6181 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6241 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6301 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6361 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6421 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6481 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6541 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6601 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6661 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     6721 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     6781 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     6841 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcag
//
