LOCUS       sequence-390988-        7574 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To study u-crystallin protein interactors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7574)
  AUTHORS   Ollivier M, Soto JS, Linker KE, Moye SL, Jami-Alahmadi Y, Jones AE, 
            Divakaruni AS, Kawaguchi R, Wohlschlegel JA, Khakh BS
  TITLE     Crym-positive striatal astrocytes gate perseverative behaviour.
  JOURNAL   Nature. 2024 Mar;627(8003):358-366. doi: 10.1038/s41586-024-07138-0.
            Epub 2024 Feb 28.
  PUBMED    38418885
REFERENCE   2  (bases 1 to 7574)
  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: 
            "10.1038/s41586-024-07138-0"; journalName: "Nature"; date: 
            "2024-03"; volume: "627"; issue: "8003"; pages: "358-366"
COMMENT     Sequence Label: GfaABC1D-Crym-BioID2-BioID2
FEATURES             Location/Qualifiers
     source          1..7574
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   259..388
                     /label=AAV2 ITR
     intron          1239..1371
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1354..1373
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1416..1434
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2405..3103
                     /codon_start=1
                     /product="promiscuous R40G mutant of a biotin protein 
                     ligase from Aquifex aeolicus (Kim et al., 2016)"
                     /label=BioID2
                     /translation="MFKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKW
                     LSQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKV
                     SGVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISE
                     NLKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKE
                     ILSGEFSLRRS"
     CDS             3188..3886
                     /codon_start=1
                     /product="promiscuous R40G mutant of a biotin protein 
                     ligase from Aquifex aeolicus (Kim et al., 2016)"
                     /label=BioID2
                     /translation="MFKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKW
                     LSQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKV
                     SGVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISE
                     NLKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKE
                     ILSGEFSLRRS"
     CDS             3896..3922
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    3947..4535
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4000..4020)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4418..4429)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4599..4720
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4610..4629)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4664..4683
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4882..5011
                     /label=AAV2 ITR
     primer_bind     complement(5030..5047)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5030..5046)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5039..5061)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5188..5643
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5275..5294)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5485..5506
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5669..5773
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5774..5842,5843..6634)
                     /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"
     rep_origin      6805..7393
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7294..7313
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
      241 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatctaa catatcctgg tgtggagtag gggacgctgc
      481 tctgacagag gctcgggggc ctgagctggc tctgtgagct ggggaggagg cagacagcca
      541 ggccttgtct gcaagcagac ctggcagcat tgggctggcc gccccccagg gcctcctctt
      601 catgcccagt gaatgactca ccttggcaca gacacaatgt tcggggtggg cacagtgcct
      661 gcttcccgcc gcaccccagc ccccctcaaa tgccttccga gaagcccatt gagcaggggg
      721 cttgcattgc accccagcct gacagcctgg catcttggga taaaagcagc acagccccct
      781 aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga acaaggctct attcagcctg
      841 tgcccaggaa aggggatcag gggatgccca ggcatggaca gtgggtggca gggggggaga
      901 ggagggctgt ctgcttccca gaagtccaag gacacaaatg ggtgagggga gagctctccc
      961 catagctggg ctgcggccca accccacccc ctcaggctat gccagggggt gttgccaggg
     1021 gcacccgggc atcgccagtc tagcccactc cttcataaag ccctcgcatc ccaggagcga
     1081 gcagagccag agcaggttgg agaggagacg catcacctcc gctgctcgca agctttattg
     1141 cggtagttta tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga
     1201 acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac
     1261 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct
     1321 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact
     1381 cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta taggctagcc
     1441 tcgaatgaag cgggcgccag cgttcctgag cgcagaggag gtgcaggatc accttcgcag
     1501 ctccagcctt ctcatcccac ccctggaggc cgcactggcc aacttctcca aaggtcccga
     1561 cggaggggtc atgcagccag tgcgcaccgt ggtgcctgta gccaagcacc gaggcttcct
     1621 gggagtcatg cctgcctaca gtgctgctga ggatgcgctc accaccaagt tagtcacctt
     1681 ctatgagggc cacagcaaca cagcggtccc ctcccatcag gcatcggtgc ttctctttga
     1741 tcccagcaat ggctccctgc tggcggtcat ggatggaaat gtcataactg caaagagaac
     1801 agcagcggtg tctgccattg ccacaaagct gttgaagccc ccaggcagtg atgtgctgtg
     1861 catccttgga gcgggggtcc aggcgtacag tcactatgag atcttcacag agcagttctc
     1921 cttcaaggag gtgagaatgt ggaaccgcac cagggaaaat gctgagaagt ttgcaagcac
     1981 agtgcaagga gatgttcggg tctgttcatc agtgcaggag gctgtgacag gtgctgatgt
     2041 catcatcaca gtcaccatgg caacagagcc cattttattt ggtgaatggg taaagccggg
     2101 ggctcacatc aatgctgttg gagccagcag gcctgactgg cgagaactgg atgacgagct
     2161 catgaggcaa gcggtgctgt atgtggactc ccgggaggct gccctgaagg agtcaggaga
     2221 cgttctgttg tcaggggctg acatctttgc tgagcttgga gaagtgattt caggagcgaa
     2281 gcctgcacac tgtgagaaga ccacagtgtt caaatctttg gggatggcag tggaagacct
     2341 ggttgcagcc aaattagtat atgattcttg gtcatctggc aagatcgagc tcggatccac
     2401 caccatgttc aaaaatctta tttggctgaa ggaagttgat agcacccagg agcggcttaa
     2461 ggaatggaat gtctcctatg gcactgcact cgtcgccgat agacagacca aaggtagagg
     2521 cggtctcgga cggaagtggt tgagtcagga gggcggtctt tacttctctt tcttgttgaa
     2581 tccaaaagag tttgaaaatt tgttgcaact tccactggtc ttgggactct ccgtgagcga
     2641 ggcactcgaa gagattacag agataccatt tagtctgaag tggcccaacg acgtttattt
     2701 tcaggagaaa aaggtttcag gggtcctttg tgagctcagt aaggacaagc tgatagtggg
     2761 aatcggtata aacgtgaatc aaagagagat ccctgaagag atcaaggatc gggccacaac
     2821 actttatgaa atcactggaa aggattggga cagaaaggag gtcttgctga aggttctcaa
     2881 gcgaatttcc gaaaatctca agaaatttaa ggaaaaatcc tttaaagagt tcaaaggaaa
     2941 aattgagtca aagatgttgt accttggaga ggaggttaaa ctcttgggag aaggcaagat
     3001 taccgggaaa cttgtgggcc tgagtgaaaa gggcggtgct ttgatactta cagaggaagg
     3061 cataaaggag atactgtccg gcgaatttag cttgcgacgg tcaagcttag gtggaggagg
     3121 ttctggaggc ggtggaagtg gtggcggagg tagcggtgga ggaggttcta tggtgtctaa
     3181 ggaattcatg ttcaaaaatc ttatttggct gaaggaagtt gatagcaccc aggagcggct
     3241 taaggaatgg aatgtctcct atggcactgc actcgtcgcc gatagacaga ccaaaggtag
     3301 aggcggtctc ggacggaagt ggttgagtca ggagggcggt ctttacttct ctttcttgtt
     3361 gaatccaaaa gagtttgaaa atttgttgca acttccactg gtcttgggac tctccgtgag
     3421 cgaggcactc gaagagatta cagagatacc atttagtctg aagtggccca acgacgttta
     3481 ttttcaggag aaaaaggttt caggggtcct ttgtgagctc agtaaggaca agctgatagt
     3541 gggaatcggt ataaacgtga atcaaagaga gatccctgaa gagatcaagg atcgggccac
     3601 aacactttat gaaatcactg gaaaggattg ggacagaaag gaggtcttgc tgaaggttct
     3661 caagcgaatt tccgaaaatc tcaagaaatt taaggaaaaa tcctttaaag agttcaaagg
     3721 aaaaattgag tcaaagatgt tgtaccttgg agaggaggtt aaactcttgg gagaaggcaa
     3781 gattaccggg aaacttgtgg gcctgagtga aaagggcggt gctttgatac ttacagagga
     3841 aggcataaag gagatactgt ccggcgaatt tagcttgcga cggtcagcta gcgcttaccc
     3901 ttacgatgta ccggattacg catagggatc caattcgctt atcgataatc aacctctgga
     3961 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     4021 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     4081 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     4141 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     4201 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     4261 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     4321 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct atgttgccac
     4381 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     4441 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     4501 gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccggaatt ctgcagatat
     4561 ctagagtcga cccgggcggc cgcttcgagc agacatgata agatacattg atgagtttgg
     4621 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     4681 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     4741 ttttatgttt caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta
     4801 caaatgtggt aaaatcgata aggatcttcc tagagcatgg ctacgtagat aagtagcatg
     4861 gcgggttaat cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc
     4921 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     4981 gggcggcctc agtgagcgag cgagcgcgca gccttaatta acctaattca ctggccgtcg
     5041 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     5101 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     5161 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     5221 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     5281 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     5341 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     5401 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5461 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     5521 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     5581 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     5641 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5701 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5761 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5821 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5881 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5941 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     6001 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     6061 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     6121 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     6181 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     6241 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6301 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6361 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6421 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6481 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6541 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6601 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6661 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6721 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6781 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     6841 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     6901 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     6961 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     7021 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     7081 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     7141 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     7201 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     7261 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     7321 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     7381 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     7441 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     7501 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     7561 cgaggaagcg gaag
//
