LOCUS       sequence-318948-        6311 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Reporter for activity of the CLEAR network.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6311)
  AUTHORS   Cortes CJ, Miranda HC, Frankowski H, Batlevi Y, Young JE, Le A, 
            Ivanov N, Sopher BL, Carromeu C, Muotri AR, Garden GA, La Spada AR
  TITLE     Polyglutamine-expanded androgen receptor interferes with TFEB to 
            elicit autophagy defects in SBMA.
  JOURNAL   Nat Neurosci. 2014 Sep;17(9):1180-9. doi: 10.1038/nn.3787. Epub 2014
            Aug 10.
  PUBMED    25108912
REFERENCE   2  (bases 1 to 6311)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nn.3787"; 
            journalName: "Nat Neurosci"; date: "2014-09"; volume: "17"; issue: 
            "9"; pages: "1180-9"
COMMENT     Sequence Label: 4XCLEAR-luciferase reporter
FEATURES             Location/Qualifiers
     source          1..6311
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    94..115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(130..135,136..153,154..160)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    168..184
                     /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)."
     primer_bind     173..195
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     192..208
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     192..208
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        337..439
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             440..1099
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(506..525)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     regulatory      1325..1334
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1331..2983
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     primer_bind     complement(1397..1415)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     intron          3092..3157
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             3287..3307
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3732..3866
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3769..3788)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3823..3842
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3903..3920)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3903..3919)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3912..3934)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4128..4147)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4307..4325)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4393..4497
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4498..4566,4567..5358)
                     /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(4716..4735)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5529..6117
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6018..6037
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6271..6288
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
       61 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
      121 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
      181 acaatttcac acaggaaaca gctatgacat gattacgaat tcgagctcgc ccggggctcg
      241 agctgtgacg gaagatcact tcgcagaata aataaatcct ggtgtccctg ttgataccgg
      301 gaagccctgg gccaactttt ggcgaaaatg agacgttgat cggcacgtaa gaggttccaa
      361 ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttgt cgagattttc
      421 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
      481 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
      541 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
      601 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattacg
      661 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt
      721 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
      781 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
      841 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac
      901 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg
      961 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
     1021 cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga
     1081 gtggcagggc ggggcgtaac tcgagggtca cgtgacccca gggtcacgtg accctgcggg
     1141 tcacgtgacc ctgcgggtca cgtgaccaga tctgacttct agagatccgg caaaccccgc
     1201 ccagcgtctt gtcattggcg aattcgaaca cgcagatgca gtcggggcgg cgcggtccga
     1261 ggtccacttc gcatattaag gtgacgcgtg tggcctcgaa caccgagcga ccctgcataa
     1321 gcttgccacc atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatcctct
     1381 agaggatgga accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc
     1441 tggaacaatt gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt
     1501 cgaaatgtcc gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag
     1561 aatcgtcgta tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt
     1621 tatcggagtt gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag
     1681 tatgaacatt tcgcagccta ccgtagtgtt tgtttccaaa aaggggttgc aaaaaatttt
     1741 gaacgtgcaa aaaaaattac caataatcca gaaaattatt atcatggatt ctaaaacgga
     1801 ttaccaggga tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa
     1861 tgaatacgat tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa
     1921 ttcctctgga tctactgggt tacctaaggg tgtggccctt ccgcatagaa ctgcctgcgt
     1981 cagattctcg catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat
     2041 tttaagtgtt gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat
     2101 atgtggattt cgagtcgtct taatgtatag atttgaagaa gagctgtttt tacgatccct
     2161 tcaggattac aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa
     2221 aagcactctg attgacaaat acgatttatc taatttacac gaaattgctt ctgggggcgc
     2281 acctctttcg aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc cagggatacg
     2341 acaaggatat gggctcactg agactacatc agctattctg attacacccg agggggatga
     2401 taaaccgggc gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga
     2461 taccgggaaa acgctgggcg ttaatcagag aggcgaatta tgtgtcagag gacctatgat
     2521 tatgtccggt tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg
     2581 gctacattct ggagacatag cttactggga cgaagacgaa cacttcttca tagttgaccg
     2641 cttgaagtct ttaattaaat acaaaggata tcaggtggcc cccgctgaat tggaatcgat
     2701 attgttacaa caccccaaca tcttcgacgc gggcgtggca ggtcttcccg acgatgacgc
     2761 cggtgaactt cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga
     2821 gatcgtggat tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt
     2881 gtttgtggac gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga
     2941 gatcctcata aaggccaaga agggcggaaa gtccaaattg taaagttaac agatcggccg
     3001 cgactctagc tagaggatct ttgtgaagga accttacttc tgtggtgtga cataattgga
     3061 caaactacct acagagattt aaagctctaa ggtaaatata aaatttttaa gtgtataatg
     3121 tgttaaacta ctgattctaa ttgtttgtgt attttagatt ccaacctatg gaactgatga
     3181 atgggagcag tggtggaatg cctttaatga ggaaaacctg ttttgctcag aagaaatgcc
     3241 atctagtgat gatgaggcta ctgctgactc tcaacattct actcctccaa aaaagaagag
     3301 aaaggtagaa gaccccaagg actttccttc agaattgcta agttttttga gtcatgctgt
     3361 gtttagtaat agaactcttg cttgctttgc tatttacacc acaaaggaaa aagctgcact
     3421 gctatacaag aaaattatgg aaaaatattc tgtaaccttt ataagtaggc ataacagtta
     3481 taatcataac atactgtttt ttcttactcc acacaggcat agagtgtctg ctattaataa
     3541 ctatgctcaa aaattgtgta cctttagctt tttaatttgt aaaggggtta ataaggaata
     3601 tttgatgtat agtgccttga ctagagatca taatcagcca taccacattt gtagaggttt
     3661 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     3721 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3781 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3841 tcaatgtatc ttatcatgtc tggatcggcc gagtttccgc gggaccccgg ccagagcttg
     3901 gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     3961 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     4021 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     4081 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     4141 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4201 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4261 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     4321 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     4381 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4441 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4501 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4561 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4621 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4681 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4741 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4801 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4861 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4921 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4981 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5041 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5101 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5161 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5221 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5281 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5341 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5401 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5461 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5521 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5581 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5641 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5701 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5761 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5821 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5881 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5941 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6001 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6061 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6121 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6181 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     6241 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     6301 cgcccaatac g
//
