LOCUS       sequence-390258-        6355 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses Human N-Terminal 3X Flag CASP9 Catalytic Mutant in 
            Mammalian Cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6355)
  AUTHORS   Han J, Goldstein LA, Hou W, Watkins SC, Rabinowich H
  TITLE     Involvement of CASP9 (caspase 9) in IGF2R/CI-MPR endosomal 
            transport.
  JOURNAL   Autophagy. 2021 Jun;17(6):1393-1409. doi: 
            10.1080/15548627.2020.1761742. Epub 2020 May 25.
  PUBMED    32397873
REFERENCE   2  (bases 1 to 6355)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1080/15548627.2020.1761742"; journalName: "Autophagy"; date: 
            "2021-06"; volume: "17"; issue: "6"; pages: "1393-1409"
COMMENT     Sequence Label: N-terminal 3X Flag Human CASP9 C287A Mutant
FEATURES             Location/Qualifiers
     source          1..6355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        10..389
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..593
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     544..564
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     638..657
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        638..656
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             716..781
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(1511..1522)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(2102..2119)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2108..2332
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2464..3052)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2544..2563)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3381..3428
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3453..3472
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3660..4454)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3660..4454)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3771..3790)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4381..4400
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4489..4846)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4503..4638
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        4655..4846
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     4803..4823
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(4906..5697,5698..5766))
                     /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     5529..5548
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5767..5871)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5898..6353
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5985..6004)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6195..6216
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
       61 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      121 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      181 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      241 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      361 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      421 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      481 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      541 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc
      601 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga
      661 cccaagctgg ctagcgttta aacttaagct tggtaccgag ctcggatccg ccatggacta
      721 caaagaccat gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa
      781 gatggacgaa gcggatcggc ggctcctgcg gcggtgccgg ctgcggctgg tggaagagct
      841 gcaggtggac cagctctggg acgccctgct gagccgcgag ctgttcaggc cccatatgat
      901 cgaggacatc cagcgggcag gctctggatc tcggcgggat caggccaggc agctgatcat
      961 agatctggag actcgaggga gtcaggctct tcctttgttc atctcctgct tagaggacac
     1021 aggccaggac atgctggctt cgtttctgcg aactaacagg caagcagcaa agttgtcgaa
     1081 gccaacccta gaaaacctta ccccagtggt gctcagacca gagattcgca aaccagaggt
     1141 tctcagaccg gaaacaccca gaccagtgga cattggttct ggaggatttg gtgatgtcgg
     1201 tgctcttgag agtttgaggg gaaatgcaga tttggcttac atcctgagca tggagccctg
     1261 tggccactgc ctcattatca acaatgtgaa cttctgccgt gagtccgggc tccgcacccg
     1321 cactggctcc aacatcgact gtgagaagtt gcggcgtcgc ttctcctcgc tgcatttcat
     1381 ggtggaggtg aagggcgacc tgactgccaa gaaaatggtg ctggctttgc tggagctggc
     1441 gcagcaggac cacggtgctc tggactgctg cgtggtggtc attctctctc acggctgtca
     1501 ggccagccac ctgcagttcc caggggctgt ctacggcaca gatggatgcc ctgtgtcggt
     1561 cgagaagatt gtgaacatct tcaatgggac cagctgcccc agcctgggag ggaagcccaa
     1621 gctctttttc atccaggccg cgggtgggga gcagaaagac catgggtttg aggtggcctc
     1681 cacttcccct gaagacgagt cccctggcag taaccccgag ccagatgcca ccccgttcca
     1741 ggaaggtttg aggaccttcg accagctgga cgccatatct agtttgccca cacccagtga
     1801 catctttgtg tcctactcta ctttcccagg ttttgtttcc tggagggacc ccaagagtgg
     1861 ctcctggtac gttgagaccc tggacgacat ctttgagcag tgggctcact ctgaagacct
     1921 gcagtccctc ctgcttaggg tcgctaatgc tgtttcggtg aaagggattt ataaacagat
     1981 gcctggttgc tttaatttcc tccggaaaaa acttttcttt aaaacatcat aaggccaggg
     2041 cccctcaccc tgccttatct tgcagtcgag tctagagggc ccgtttaaac ccgctgatca
     2101 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     2161 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     2221 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     2281 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     2341 gcggaaagaa ccagtggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2401 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2461 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2521 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2581 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2641 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2701 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2761 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2821 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2881 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     2941 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3001 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3061 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3121 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3181 taaatcaatc taaagtatat atgagtaacc tgaggctatg gcagggcctg ccgccccgac
     3241 gttggctgcg agccctgggc cttcacccga acttgggggg tggggtgggg aaaaggaaga
     3301 aacgcgggcg tattggcccc aatggggtct cggtggggta tcgacagagt gccagccctg
     3361 ggaccgaacc ccgcgtttat gaacaaacga cccaacaccg tgcgttttat tctgtctttt
     3421 tattgccgtc atagcgcggg ttccttccgg tattgtctcc ttccgtgttt cagttagcct
     3481 ccccctaggg tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc
     3541 ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa
     3601 atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct
     3661 cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata
     3721 ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg
     3781 gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat
     3841 ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg
     3901 acgagatcct cgccgtcggg catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg
     3961 agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta
     4021 cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc
     4081 gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga
     4141 gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca
     4201 gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc
     4261 gctgcctcgt cttgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc
     4321 gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt
     4381 gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca
     4441 tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcgatctt tgcaaaagcc
     4501 taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggccg aggcggcctc
     4561 ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg
     4621 cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag
     4681 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc
     4741 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     4801 acaccctaac tgacacacat tccacagctg gttctttccg cctcaggact cttccttttt
     4861 caataaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4921 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4981 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5041 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5101 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5161 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5221 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5281 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5341 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5401 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5461 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5521 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5581 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5641 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5701 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5761 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5821 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5881 ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5941 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     6001 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     6061 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     6121 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     6181 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     6241 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     6301 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttac
//
