LOCUS       sequence-434245-        7112 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CaST-P2A for HEK cell expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7112)
  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 7112)
  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-P2A-CaM-V5-sTN
FEATURES             Location/Qualifiers
     source          1..7112
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(384..440)
                     /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             complement(join(450..1160,1161..1163,1164..1166))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(450..1160,1161..1163,1164..1166))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(492..513)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     841..860
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             complement(1197..1253)
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     CDS             complement(2778..2804)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2805..2831)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     sig_peptide     complement(2856..2915)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     regulatory      2912..2921
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2934..2953)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2935..2953)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2998..3201)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3027..3047)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3202..3505
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   3576..3705
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(3767..4355)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3847..3866)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4526..5317,5318..5386))
                     /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"
     promoter        complement(5387..5491)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5559..5577
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5615..5637)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5737..5756
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5773..6228)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5910..5931)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6122..6141
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6303..6443
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6314..6443
                     /label=AAV2 ITR
     polyA_signal    6472..6679
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6668..6685
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(6944..6985)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
ORIGIN
        1 tctgcttccc tgatcatttc atcaacctct tcatctgtta acttctctcc aaggtttgtc
       61 atcacgtggc gaagctctgc tgcactgatg tagccattgc catccttatc aaacacaccg
      121 aacgcttctc taatttcttc ttccgtgtcc ctgtatttca tttttcttgc catcattgtc
      181 aggaactcag ggaagtcgat tgtgccgtca ccgtcggcat ctacttcatt gatcatgtcc
      241 tgcagctctg cttctgtggg gttctgcccc agagaccgca tcaccgtccc cagctccttg
      301 gttgttattg tcccatcccc gtccttgtca aatagggaga aagcctcttt aaattctgcg
      361 atctgctctt cagtcagttg gtctggcccg ggattctctt cgacatcccc tgcttgtttc
      421 aacagggaga agttagtggc tccgcttccc ttgtacagct cgtccatgcc gagagtgatc
      481 ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg
      541 ctcagggcgg actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg
      601 atgggggtgt tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg
      661 cggatcttga agttcacctt gatgccgttc ttctgcttgt cggccatgat atagacgttg
      721 tggctgttgt agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg
      781 atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg
      841 gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc
      901 atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc
      961 acgccgtagg tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg
     1021 cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg
     1081 ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg
     1141 aacagctcct cgcccttgct caccatagat ccaccgctgc ctgatccact tccacctgag
     1201 ctcagccgac ctatagctct gactgcgtga cctgtcttat tccacttacg acgaccgctc
     1261 ccactaccac cggtactccc gcccgagccc ttttcggcag accgcagact gatttctccg
     1321 cccatccagg gtttgataac tccgtcctgt tccagcagca gggctccctg tttatcaatc
     1381 ccccggctaa tcccgaaaat ctctttgtcc ccaatgatca gcttcacggg tctattgatg
     1441 aagttatcca gcttctccca ccgtggcaga tatggagcca ggccttcctg ctcgaacagt
     1501 tccagggctg cccgcagctc tcggatcagc gtagcggcca gagtattcct gtccaggtta
     1561 atccctgctt cctgcagtgt gatccagccc tgattgacca cgctttcctc cacgcgcctc
     1621 atagccacgt taatcccggc gccaatgacg atctgtgcag catcgcctgt tattccggcc
     1681 agctccacca ggatgcctgc cagctttcta tcctgcagat acaggtcatt gggccatttg
     1741 actcgcacct tgtctgctcc cagctttcgc agcgcttctg ccatgacaat tccgatgacc
     1801 gggcccaggc cgattgctgc tggtccccgc ttcaggcgcc aaaacatact caggtacagg
     1861 ttagctccaa aaggagagaa ccatttcctc cctctgcttc ctctccctgc ctgctgatat
     1921 tctgcaatgc aagcatcccc actcttcagc tcgccgattc gatccagcag gtactgattg
     1981 gtggagtcga ccacaggcag gactgccacg ctcccgccgt ccagctgtcc acctccagag
     2041 ctgccaccag aacctctaga gccaccgctg cccgagcccg aggtcgagcc cgagcccttg
     2101 cgccttcggt gccggcacct gacacagaag aagatgccta gcccaatgaa aagcaggagg
     2161 ccggcgacgc cccccagcac aatcagggcc attggctgca ccggggtgga ccatgtgggc
     2221 agaaccttga tgttggattc cagcaggacc tgtcccgagt cactcagcag acactgccac
     2281 atccccgcct cagggttcag cacccacacc gccttctccc gcttcgagac ctttgcctcc
     2341 ttgttctcca gtttcaagct cagcatcagc ttaggggagg tgggtcccca cacctcacag
     2401 gtcaaatttt tctggagctg agtggctctc atcaccacca ggttcacttc ctgatgcaac
     2461 tttcctgttt tcgcttcaag ggccagggtg aggtttccag agccagcata ctgaggcaag
     2521 gcctggggca gggtgaggtg gagcgggagc ttcttgccca tctggagctt agggtcctgg
     2581 gtaacccgtt ttacagacac ttccttgttc ttcaggtcaa aggtgatcca agacttggag
     2641 gaggaagccc tctccgcctg ccaccacagc tcgccactgc ccgtcagctt ttcaactgta
     2701 aaggcgagtg ggaaggagaa ctccacctgt tccccctctt tcttatagac tatgctggag
     2761 gccttctgga agtcgagagc gtaatcaggg acgtcatagg gataggcgta atctggcaca
     2821 tcgtatgggt aagatctggc cggctgggcc ccgtcaccag tggaacctgg aacccagagc
     2881 agcagtaccc atagcaggag tgtgtctgtc tccatggtgg caagcttggg tctccctata
     2941 gtgagtcgta ttaatttcga taagccagta agcagtgggt tctctagtta gccagagagc
     3001 tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca atggggcgga
     3061 gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac tcccattgac
     3121 gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac gcccattgat
     3181 gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga tgtactgcca
     3241 agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc
     3301 attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc
     3361 agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg gcgttactat
     3421 gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg
     3481 gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa ctaatgaccc
     3541 cgtaattgat tactatgtac aacgcgtgcg gccgcaggaa cccctagtga tggagttggc
     3601 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     3661 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggacatg tgagcaaaag
     3721 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3781 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3841 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3901 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3961 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4021 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4081 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4141 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4201 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4261 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4321 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4381 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4441 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4501 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4561 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4621 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcggctt ccacgctcac
     4681 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4741 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4801 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4861 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4921 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4981 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5041 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5101 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5161 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5221 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5281 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5341 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5401 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5461 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     5521 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     5581 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     5641 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     5701 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     5761 gagtgcacca taaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt
     5821 aaatcagctc attttttaac caatagaccg aaatcggcaa aatcccttat aaatcaaaag
     5881 aatagcccga gatagagttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     5941 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     6001 aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     6061 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     6121 aagggaagaa agcgaaagga gcgggcgcta aggcgctggc aagtgtagcg gtcacgctgc
     6181 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtac tatggttgct
     6241 ttgacgtatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     6301 cccctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg
     6361 tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc
     6421 caactccatc actaggggtt cctgcggccg ctcggtccgc acgtggttac ctccccagca
     6481 tgcctgctat tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga
     6541 atagaatgac acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca
     6601 gtgggagtgg caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc
     6661 tggcaactag aaggcacagt cgaggctgat cagcgagctc tagctcgagg aattcttatt
     6721 acagaatctg tttagcgttc agcagcggga taggctcggg cagagagtag ccctttccgg
     6781 gcacggtgaa cacgtccacg ccccagtcgc gcagagtctg aatgtgcttg ttgatagcgg
     6841 ccctggacat gcccagggtt tctcccagct gttcgccact atggaactcg ccattagcca
     6901 ggagagcgat cagcttcaga ggcacagtat tgtctttgct agcggtgctg tccaggccca
     6961 gcagggggtt ggggatgggc ttgccacctc cagagctgcc accagaacct ccagagccac
     7021 cgctgcccga gcccgaggtc gagcccgagc ccttcttcgc tgtcatcatt tgtacaaact
     7081 cttcgtagtt tacctgacca tccccatcga tg
//
