LOCUS       sequence-371188-        5973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the small actin binding peptide, LifeAct, with a GFP 
            reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5973)
  AUTHORS   Soto JS, Jami-Alahmadi Y, Chacon J, Moye SL, Diaz-Castro B, 
            Wohlschlegel JA, Khakh BS
  TITLE     Astrocyte-neuron subproteomes and obsessive-compulsive disorder 
            mechanisms.
  JOURNAL   Nature. 2023 Apr;616(7958):764-773. doi: 10.1038/s41586-023-05927-7.
            Epub 2023 Apr 12.
  PUBMED    37046092
REFERENCE   2  (bases 1 to 5973)
  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/s41586-023-05927-7"; journalName: "Nature"; date: 
            "2023-04"; volume: "616"; issue: "7958"; pages: "764-773"
COMMENT     Sequence Label: pZac2.1  gfaABC1D-LifeAct-GFP
FEATURES             Location/Qualifiers
     source          1..5973
                     /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             1455..1505
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     regulatory      1521..1530
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1527..1529,1530..1532,1533..2243)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1572..1593)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1833..1852)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2244..2309
                     /label=MCS
                     /note="multiple cloning site"
     misc_feature    2346..2934
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2399..2419)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2817..2828)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2998..3119
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3009..3028)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3063..3082
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3429..3446)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3429..3445)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3438..3460)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3587..4042
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3674..3693)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3884..3905
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4068..4172
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4173..4241,4242..5033)
                     /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      5204..5792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5693..5712
                     /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 tcgagctcgg atccatgggc gtggccgacc tgatcaagaa gttcgagagc atcagcaagg
     1501 aagagggcga cccaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     1561 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1621 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1681 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1741 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1801 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1861 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1921 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     1981 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2041 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2101 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     2161 ccaacgagaa gcgcgatcac atggtcctta aggagttcgt gaccgccgcc gggatcactc
     2221 tcggcatgga cgagctgtac aagtccggac tcagatctcg agctcaagct tcgaattctg
     2281 cagtcgacgg taccgcgggc ccgggatcca ccggatctag ataaggatcc aattcgctta
     2341 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     2401 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     2461 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     2521 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     2581 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     2641 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     2701 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     2761 tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     2821 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     2881 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     2941 accggaattc tgcagatatc tagagtcgac ccgggcggcc gcttcgagca gacatgataa
     3001 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     3061 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     3121 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt
     3181 aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatcttcct agagcatggc
     3241 tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag
     3301 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     3361 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcrg ccttaattaa
     3421 cctaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     3481 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     3541 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     3601 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3661 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3721 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3781 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3841 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3901 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3961 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4021 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4081 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4141 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     4201 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     4261 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     4321 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     4381 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     4441 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     4501 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     4561 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     4621 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     4681 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     4741 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     4801 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     4861 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     4921 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     4981 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     5041 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     5101 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     5161 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5221 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     5281 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5341 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5401 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5461 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5521 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5581 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5641 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5701 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5761 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5821 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5881 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     5941 agcgcagcga gtcagtgagc gaggaagcgg aag
//
