LOCUS       sequence-434257-        7631 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CaST-IRES for HEK cell expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7631)
  AUTHORS   Zhang R, Anguiano M, Aarrestad IK, Lin S, Chandra J, Vadde SS, Olson
            DE, Kim CK
  TITLE     Rapid, biochemical tagging of cellular activity history in vivo.
  JOURNAL   Nat Methods. 2024 Sep;21(9):1725-1735. doi: 
            10.1038/s41592-024-02375-7. Epub 2024 Aug 5.
  PUBMED    39103446
REFERENCE   2  (bases 1 to 7631)
  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/s41592-024-02375-7"; journalName: "Nat Methods"; date: 
            "2024-09"; volume: "21"; issue: "9"; pages: "1725-1735"
COMMENT     Sequence Label: pAAV-CMV-CD4-sTC-M13-GFP-IRES-CaM-V5-sTN
FEATURES             Location/Qualifiers
     source          1..7631
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      18..27
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             537..578
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(837..854)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    843..1050
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   1079..1219
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1079..1208
                     /label=AAV2 ITR
     rep_origin      1294..1749
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1381..1400)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1591..1612
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1766..1785)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1885..1907
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1945..1963)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2031..2135
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2136..2204,2205..2996)
                     /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      3167..3755
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3656..3675
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   3817..3946
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        4017..4320
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4321..4524
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4475..4495
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4569..4588
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4569..4587
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4601..4610
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     4607..4666
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             4691..4717
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4718..4744
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6269..6325
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     CDS             join(6356..6358,6359..6361,6362..7075)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6356..6358,6359..6361,6362..7072)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6662..6681)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7009..7030
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(7088..7631,1..9)
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(7232..7249)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     7459..7478
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
ORIGIN
        1 atgataatat ggccacagcc accatggacc aactgactga agagcagatc gcagaattta
       61 aagaggcttt ctccctattt gacaaggacg gggatgggac aataacaacc aaggagctgg
      121 ggacggtgat gcggtctctg gggcagaacc ccacagaagc agagctgcag gacatgatca
      181 atgaagtaga tgccgacggt gacggcacaa tcgacttccc tgagttcctg acaatgatgg
      241 caagaaaaat gaaatacagg gacacggaag aagaaattag agaagcgttc ggtgtgtttg
      301 ataaggatgg caatggctac atcagtgcag cagagcttcg ccacgtgatg acaaaccttg
      361 gagagaagtt aacagatgaa gaggttgatg aaatgatcag ggaagcagac atcgatgggg
      421 atggtcaggt aaactacgaa gagtttgtac aaatgatgac agcgaagaag ggctcgggct
      481 cgacctcggg ctcgggcagc ggtggctctg gaggttctgg tggcagctct ggaggtggca
      541 agcccatccc caaccccctg ctgggcctgg acagcaccgc tagcaaagac aatactgtgc
      601 ctctgaagct gatcgctctc ctggctaatg gcgagttcca tagtggcgaa cagctgggag
      661 aaaccctggg catgtccagg gccgctatca acaagcacat tcagactctg cgcgactggg
      721 gcgtggacgt gttcaccgtg cccggaaagg gctactctct gcccgagcct atcccgctgc
      781 tgaacgctaa acagattctg taataagaat tcctcgagct agagctcgct gatcagcctc
      841 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
      901 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
      961 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     1021 ttgggaagac aatagcaggc atgctgggga ggtaaccacg tgcggaccga gcggccgcag
     1081 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     1141 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     1201 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     1261 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     1321 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     1381 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1441 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1501 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1561 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1621 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     1681 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     1741 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     1801 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     1861 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     1921 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     1981 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     2041 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2101 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     2161 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     2221 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     2281 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     2341 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     2401 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     2461 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     2521 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     2581 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     2641 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     2701 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     2761 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     2821 ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc
     2881 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     2941 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     3001 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3061 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3121 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3181 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3241 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3301 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3361 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     3421 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3481 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3541 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3601 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3661 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3721 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3781 ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct
     3841 cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     3901 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg ccgcacgcgt
     3961 tgtacatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     4021 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     4081 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     4141 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     4201 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     4261 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     4321 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     4381 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     4441 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     4501 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
     4561 atcgaaatta atacgactca ctatagggag acccaagctt gccaccatgg agacagacac
     4621 actcctgcta tgggtactgc tgctctgggt tccaggttcc actggtgacg gggcccagcc
     4681 ggccagatct tacccatacg atgtgccaga ttacgcctat ccctatgacg tccctgatta
     4741 cgctctcgac ttccagaagg cctccagcat agtctataag aaagaggggg aacaggtgga
     4801 gttctccttc ccactcgcct ttacagttga aaagctgacg ggcagtggcg agctgtggtg
     4861 gcaggcggag agggcttcct cctccaagtc ttggatcacc tttgacctga agaacaagga
     4921 agtgtctgta aaacgggtta cccaggaccc taagctccag atgggcaaga agctcccgct
     4981 ccacctcacc ctgccccagg ccttgcctca gtatgctggc tctggaaacc tcaccctggc
     5041 ccttgaagcg aaaacaggaa agttgcatca ggaagtgaac ctggtggtga tgagagccac
     5101 tcagctccag aaaaatttga cctgtgaggt gtggggaccc acctccccta agctgatgct
     5161 gagcttgaaa ctggagaaca aggaggcaaa ggtctcgaag cgggagaagg cggtgtgggt
     5221 gctgaaccct gaggcgggga tgtggcagtg tctgctgagt gactcgggac aggtcctgct
     5281 ggaatccaac atcaaggttc tgcccacatg gtccaccccg gtgcagccaa tggccctgat
     5341 tgtgctgggg ggcgtcgccg gcctcctgct tttcattggg ctaggcatct tcttctgtgt
     5401 caggtgccgg caccgaaggc gcaagggctc gggctcgacc tcgggctcgg gcagcggtgg
     5461 ctctagaggt tctggtggca gctctggagg tggacagctg gacggcggga gcgtggcagt
     5521 cctgcctgtg gtcgactcca ccaatcagta cctgctggat cgaatcggcg agctgaagag
     5581 tggggatgct tgcattgcag aatatcagca ggcagggaga ggaagcagag ggaggaaatg
     5641 gttctctcct tttggagcta acctgtacct gagtatgttt tggcgcctga agcggggacc
     5701 agcagcaatc ggcctgggcc cggtcatcgg aattgtcatg gcagaagcgc tgcgaaagct
     5761 gggagcagac aaggtgcgag tcaaatggcc caatgacctg tatctgcagg atagaaagct
     5821 ggcaggcatc ctggtggagc tggccggaat aacaggcgat gctgcacaga tcgtcattgg
     5881 cgccgggatt aacgtggcta tgaggcgcgt ggaggaaagc gtggtcaatc agggctggat
     5941 cacactgcag gaagcaggga ttaacctgga caggaatact ctggccgcta cgctgatccg
     6001 agagctgcgg gcagccctgg aactgttcga gcaggaaggc ctggctccat atctgccacg
     6061 gtgggagaag ctggataact tcatcaatag acccgtgaag ctgatcattg gggacaaaga
     6121 gattttcggg attagccggg ggattgataa acagggagcc ctgctgctgg aacaggacgg
     6181 agttatcaaa ccctggatgg gcggagaaat cagtctgcgg tctgccgaaa agggctcggg
     6241 cgggagtacc ggtggtagtg ggagcggtcg tcgtaagtgg aataagacag gtcacgcagt
     6301 cagagctata ggtcggctga gctcaggtgg aagtggatca ggcagcggtg gatctatggt
     6361 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     6421 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     6481 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     6541 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     6601 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     6661 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     6721 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     6781 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     6841 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     6901 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     6961 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     7021 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtagccccc
     7081 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
     7141 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
     7201 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg
     7261 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     7321 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     7381 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     7441 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     7501 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt
     7561 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
     7621 tgaaaaacac g
//
