LOCUS       sequence-434243-        6272 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sTb(C) half CaST for HEK cell expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6272)
  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 6272)
  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-sTb(C)-M13-GFP
FEATURES             Location/Qualifiers
     source          1..6272
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   80..209
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        280..583
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        584..787
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     738..758
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     832..851
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        832..850
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      864..873
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     870..929
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             954..980
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             981..1007
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2532..2588
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     CDS             join(2619..2621,2622..2624,2625..3338)
                     /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(2619..2621,2622..2624,2625..3335)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2925..2944)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3272..3293
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3372..3389)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3378..3585
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3614..3754
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3614..3743
                     /label=AAV2 ITR
     rep_origin      3829..4284
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3916..3935)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4126..4147
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4301..4320)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4420..4442
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4480..4498)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4566..4670
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4671..4739,4740..5531)
                     /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      join(5702..6272,1..18)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6191..6210
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
       61 tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc
      121 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
      181 agtggccaac tccatcacta ggggttcctg cggccgcacg cgttgtacat agtaatcaat
      241 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      301 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      361 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      421 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      481 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      541 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
      601 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
      661 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
      721 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      781 cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac
      841 tcactatagg gagacccaag cttgccacca tggagacaga cacactcctg ctatgggtac
      901 tgctgctctg ggttccaggt tccactggtg acggggccca gccggccaga tcttacccat
      961 acgatgtgcc agattacgcc tatccctatg acgtccctga ttacgctctc gacttccaga
     1021 aggcctccag catagtctat aagaaagagg gggaacaggt ggagttctcc ttcccactcg
     1081 cctttacagt tgaaaagctg acgggcagtg gcgagctgtg gtggcaggcg gagagggctt
     1141 cctcctccaa gtcttggatc acctttgacc tgaagaacaa ggaagtgtct gtaaaacggg
     1201 ttacccagga ccctaagctc cagatgggca agaagctccc gctccacctc accctgcccc
     1261 aggccttgcc tcagtatgct ggctctggaa acctcaccct ggcccttgaa gcgaaaacag
     1321 gaaagttgca tcaggaagtg aacctggtgg tgatgagagc cactcagctc cagaaaaatt
     1381 tgacctgtga ggtgtgggga cccacctccc ctaagctgat gctgagcttg aaactggaga
     1441 acaaggaggc aaaggtctcg aagcgggaga aggcggtgtg ggtgctgaac cctgaggcgg
     1501 ggatgtggca gtgtctgctg agtgactcgg gacaggtcct gctggaatcc aacatcaagg
     1561 ttctgcccac atggtccacc ccggtgcagc caatggccct gattgtgctg gggggcgtcg
     1621 ccggcctcct gcttttcatt gggctaggca tcttcttctg tgtcaggtgc cggcaccgaa
     1681 ggcgcaaggg ctcgggctcg acctcgggct cgggcagcgg tggctctaga ggttctggtg
     1741 gcagctctgg aggtggacag ctggacggcg ggagcgtggc agtcctgcct gtggtcgact
     1801 ccaccaatca gtacctgctg gatcgaatcg gcgagctgaa gagtggggat gcttgcattg
     1861 cagaatatca gcaggcaggg agaggaagca gagggaggaa atggttctct ccttttggag
     1921 ctaacctgta cctgagtatg ttttggcgcc tgaagcgggg accagcagca atcggcctgg
     1981 gcccggtcat cggaattgtc atggcagaag cgctgcgaaa gctgggagca gacaaggtgc
     2041 gagtcaaatg gcccaatgac ctgtatctgc aggatagaaa gctggcaggc atcctggtgg
     2101 agctggccgg aataacaggc gatgctgcac agatcgtcat tggcgccggg attaacgtgg
     2161 ctatgaggcg cgtggaggaa agcgtggtca atcagggctg gatcacactg caggaagcag
     2221 ggattaacct ggacaggaat actctggccg ctacgctgat ccgagagctg cgggcagccc
     2281 tggaactgtt cgagcaggaa ggcctggctc catatctgcc acggtgggag aagctggata
     2341 acttcatcaa tagacccgtg aagctgatca ttggggacaa agagattttc gggattagcc
     2401 gggggattga taaacaggga gccctgctgc tggaacagga cggagttatc aaaccctgga
     2461 tgggcggaga aatcagtctg cggtctgccg aaaagggctc gggcgggagt accggtggta
     2521 gtgggagcgg tcgtcgtaag tggaataaga caggtcacgc agtcagagct ataggtcggc
     2581 tgagctcagg tggaagtgga tcaggcagcg gtggatctat ggtgagcaag ggcgaggagc
     2641 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     2701 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     2761 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     2821 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2881 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     2941 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     3001 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     3061 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     3121 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     3181 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     3241 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     3301 ccgggatcac tctcggcatg gacgagctgt acaagtaata agaattcctc gagctagagc
     3361 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3421 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3481 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3541 cagcaagggg gaggattggg aagacaatag caggcatgct ggggaggtaa ccacgtgcgg
     3601 accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     3661 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     3721 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     3781 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     3841 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3901 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3961 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4021 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4081 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4141 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     4201 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4261 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     4321 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4381 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4441 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     4501 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     4561 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4621 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4681 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4741 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4801 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4861 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4921 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4981 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5041 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5101 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5161 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5221 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5281 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     5341 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca
     5401 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     5461 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     5521 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     5581 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5641 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5701 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5761 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5821 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5881 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5941 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6001 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6061 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6121 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6181 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6241 ttgagcgtcg atttttgtga tgctcgtcag gg
//
