LOCUS       sequence-320941-        5747 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FLiCRE protease for HEK293T expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5747)
  AUTHORS   Kim CK, Sanchez MI, Hoerbelt P, Fenno LE, Malenka RC, Deisseroth K, 
            Ting AY
  TITLE     A Molecular Calcium Integrator Reveals a Striatal Cell Type Driving 
            Aversion.
  JOURNAL   Cell. 2020 Dec 23;183(7):2003-2019.e16. doi: 
            10.1016/j.cell.2020.11.015. Epub 2020 Dec 11.
  PUBMED    33308478
REFERENCE   2  (bases 1 to 5747)
  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: 
            "10.1016/j.cell.2020.11.015"; journalName: "Cell"; date: 
            "2020-12-23- 23"; volume: "183"; issue: "7"; pages: "2003-2019.e16"
COMMENT     Sequence Label: CMV-GFP-CaM-uTEVp
FEATURES             Location/Qualifiers
     source          1..5747
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    148..355
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     344..361
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1100..1141)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1142..1585)
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="MDQLTEEQIAEFKEAFSLLDKDGDGTITTKELGTGMRSLGQNPTE
                     AELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGNGYISAAE
                     LRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             complement(join(1622..2332,2333..2335,2336..2338))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1622..2332,2333..2335,2336..2338))
                     /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(1664..1685)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2013..2032
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2272..2293
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2335..2344
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2357..2376)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2358..2376)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2421..2624)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2450..2470)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2625..2928
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   2999..3128
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(3190..3778)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3270..3289)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3949..4740,4741..4809))
                     /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"
     primer_bind     4572..4591
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4810..4914)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4982..5000
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5038..5060)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5160..5179
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5196..5651)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5333..5354)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5545..5564
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   join(5726..5747,1..119)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(5737..5747,1..119)
                     /label=AAV2 ITR
ORIGIN
        1 ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc
       61 ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg
      121 cggccgctcg gtccgcacgt ggttacctcc ccagcatgcc tgctattgtc ttcccaatcc
      181 tcccccttgc tgtcctgccc caccccaccc cccagaatag aatgacacct actcagacaa
      241 tgcgatgcaa tttcctcatt ttattaggaa aggacagtgg gagtggcacc ttccagggtc
      301 aaggaaggca cgggggaggg gcaaacaaca gatggctggc aactagaagg cacagtcgag
      361 gctgatcagc gagctctagc tcgaggaatt cttacaccat gaaaacttta tggccccccc
      421 acaatactga gtcagcattt aatcgccaac cactaaccca ctgctgcgcc tcctgatttg
      481 tcaacaattc catgaagttt ttcggcacgc ttgtgaaata attgtttgtg ttggtgaaat
      541 tcgatgctga gtgtatacca acaatgaacc catctctagt tgatactaat ggattgccac
      601 actgcccatc cttggtttga atccaatgct tccagaatat gccatcagat gaagggaatg
      661 tgcaactagt gtctgacacc atgctagaca tgctcttagt ttggaagttg gttgtcacaa
      721 gacatatgcg ctcttccctt tgtggctctc taaatttcag cttttgagga aatggtggga
      781 aatccttagg catgcgaata attatcatgt ccctcccatc aatgaggtgt tgttgcaaag
      841 tcgtggtgtt cttgaccttg aatacaccat gtagtgattg gaccaacagt gttccattat
      901 ttcttctaaa caagtgcttg tttgtaatga tgaagggacc aaatccaata ccatacaacg
      961 atgttgtgtg cccatcagat tcattcgtca aatgacaaat ggtgctcgat atcgggttgt
     1021 aatcacgtgg tcccttaaac aaggattctc ccccggtacc gccaccccca gatccaccgc
     1081 tgcctgatcc acttccacca gtgctgtcca ggcccagcag agggttaggg atcggcttgc
     1141 ccttcgctgt catcatttgt acaaactctt cgtagtttac ctgaccatcc ccatcgatgt
     1201 ctgcttccct gatcatttca tcaacctctt catctgttaa cttctctcca aggtttgtca
     1261 tcacgtggcg aagctctgct gcactgatgt agccattgcc atccttatca aacacacgga
     1321 acgcttctct aatttcttct tcactgtctg tgtatttcat ttttcttgcc atcattgtca
     1381 ggaactcagg gaagtcgatt gtgccgtcac cgtcggcatc tacttcattg atcatgtcct
     1441 gcagctctgc ttctgtgggg ttctgcccca gagaccgcat ccccgtcccc agctccttgg
     1501 ttgttattgt cccatccccg tccttgtcca gtagggagaa agcctcttta aattctgcga
     1561 tctgctcttc agtcagttgg tccatgcctg aaccggatcc gcctgtgcca gatccaaggt
     1621 ccttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga
     1681 ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt
     1741 ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg
     1801 cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt
     1861 tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt
     1921 gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca
     1981 gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga
     2041 agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct
     2101 tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg
     2161 tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt
     2221 aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt
     2281 ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatgg
     2341 tggcaagctt gggtctccct atagtgagtc gtattaattt cgataagcca gtaagcagtg
     2401 ggttctctag ttagccagag agctctgctt atatagacct cccaccgtac acgcctaccg
     2461 cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg ttgattttgg
     2521 tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca
     2581 aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt aatagcgatg
     2641 actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta ctgggcataa
     2701 tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac
     2761 acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg
     2821 aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg
     2881 tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata
     2941 tatgggctat gaactaatga ccccgtaatt gattactatg tacaacgcgt gcggccgcag
     3001 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     3061 gggcgaccaa aggtcgcccg acgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     3121 cctgcaggac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3181 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3241 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3301 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3361 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3421 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3481 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3541 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3601 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3661 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3721 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3781 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3841 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3901 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3961 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4021 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4081 gataccgcgg cttccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4141 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4201 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4261 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4321 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4381 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4441 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4501 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4561 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4621 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4681 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4741 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4801 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4861 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4921 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     4981 aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct
     5041 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     5101 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc
     5161 ggcatcagag cagattgtac tgagagtgca ccacaaaatt gtaaacgtta atattttgtt
     5221 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaataga ccgaaatcgg
     5281 caaaatccct tataaatcaa aagaatagcc cgagatagag ttgagtgttg ttccagtttg
     5341 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     5401 tcagggcgat ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg
     5461 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     5521 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctaaggcgct
     5581 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     5641 acagggcgcg tactatggtt gctttgacgt atgcggtgtg aaataccgca cagatgcgta
     5701 aggagaaaat accgcatcag gcgcccctgc aggcagctgc gcgctcg
//
