LOCUS       sequence-261413-        7430 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dual expression of human CAD and ICAD. The two caspase cleavage 
            sites in ICAD were mutated to TEVP cleavable sequences.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7430)
  AUTHORS   Morgan CW, Diaz JE, Zeitlin SG, Gray DC, Wells JA
  TITLE     Engineered cellular gene-replacement platform for selective and 
            inducible proteolytic profiling.
  JOURNAL   Proc Natl Acad Sci U S A. 2015 Jul 7;112(27):8344-9. doi: 
            10.1073/pnas.1504141112. Epub 2015 Jun 23.
  PUBMED    26106156
REFERENCE   2  (bases 1 to 7430)
  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: 
            "10.1073/pnas.1504141112"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2015-07-7- 7"; volume: "112"; issue: "27"; pages: "8344-9"
COMMENT     Sequence Label: pETDuet_huCAD_ICADL (TevS)
FEATURES             Location/Qualifiers
     source          1..7430
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1236..1254
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1255..1279
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             1325..1348
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     RBS             1345..1353
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             1697..1717
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2036..2056
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2396..2440
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2478..2496)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2492..2539
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2576..3031
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2663..2682)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2873..2894
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3146..3937,3938..4006))
                     /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     3769..3788
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4007..4099)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4180..4768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4669..4688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4922..4939
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4954..5093
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5019..5038)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(5195..5386)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5958..7040)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7002..7021
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7041..7118)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7324..7343
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcaccatca tcaccacagc
      121 caggatccga attcgagctc ggcgcgcctg cagatgctcc agaagcccaa gagcgtgaag
      181 ctgcgggccc tgcgcagccc gaggaagttc ggcgtggctg gccggagctg ccaggaggtg
      241 ctgcgcaagg gctgtctccg cttccagctc cctgagcgcg gttcccggct gtgcctgtac
      301 gaggatggca cggagctgac ggaagattac ttccccagtg ttcccgacaa cgccgagctg
      361 gtgctgctca ccttgggcca ggcctggcag ggctatgtga gcgacatcag gcgcttcctc
      421 agtgcatttc acgagccaca ggtggggctc atccaggccg cccagcagct gctgtgtgat
      481 gagcaggccc cacagaggca gaggctgctg gctgacctcc tgcacaacgt cagccagaac
      541 atcgcggccg agacccgggc tgaggacccg ccgtggtttg aaggcttgga gtcccgattt
      601 cagagcaagt ctggctatct gagatacagc tgtgagagcc ggatccggag ttacctgagg
      661 gaggtgagct cctacccctc cacagtgggt gcggaggctc aggaggaatt cctgcgggtc
      721 ctcggctcca tgtgccagag gctccggtcc atgcagtaca atggcagcta cttcgacaga
      781 ggagccaagg gcggcagccg cctctgcaca ccggaaggct ggttctcctg ccagggtccc
      841 tttgacatgg acagctgctt atcaagacac tccatcaacc cctacagtaa cagggagagc
      901 aggatcctct tcagcacctg gaacctggat cacataatag aaaagaaacg caccatcatt
      961 cctacactgg tggaagcaat taaggaacaa gatggaagag aagtggactg ggagtatttt
     1021 tatggcctgc tttttacctc agagaaccta aaactagtgc acattgtctg ccataagaaa
     1081 accacccaca agctcaactg tgacccgagc agaatctaca aaccccagac aaggttgaag
     1141 cggaagcagc ctgtgcggaa acgccagtga gcggccgcat aatgcttaag tcgaacagaa
     1201 agtaatcgta ttgtacacgg ccgcataatc gaaattaata cgactcacta taggggaatt
     1261 gtgagcggat aacaattccc catcttagta tattagttaa gtataagaag gagatataca
     1321 tatggattac aaggatgacg acgataagga ggtgaccggg gacgccgggg taccagaatc
     1381 tggcgagatc cggactctaa agccgtgtct gctgcgccgc aactacagcc gcgaacagca
     1441 cggcgtggcc gcctcctgcc tcgaagacct gaggagcaag gcctgtgaca ttctggccat
     1501 tgataagtcc ctgacaccag tcaccctggt cctggcagag gatggcacca tagtcgacga
     1561 cgatgactac tttctgtgtc taccttccaa tactaagttt gtggcattgg ctagtaatga
     1621 gaaatgggca tacaacaatt cagatggagg tacagcttgg atttcccaag agtcctttga
     1681 tgtagatgaa acagacgaaa atctctactt ccagagcggg gcaggattaa aatggaaaaa
     1741 cgtggccagg cagctgaaag aagatctgtc cagcatcatc ctcctatcag aggaggacct
     1801 ccagatgctt gttgacgctc cctgctcaga cctggctcag gaactacgtc agagttgtgc
     1861 caccgtccag cggctgcagc acacactcca acaggtgctt gaccaaagag aggaagtgcg
     1921 tcagtccaag cagctcctgc agctgtacct ccaggctttg gagaaagagg gcagcctctt
     1981 gtcaaagcag gaagagtcca aagctgcctt tggtgaggag gtggatgcag tagacgaaaa
     2041 tctctacttc cagagtggta cgggtatcag cagagagacc tcctcggacg ttgcgctggc
     2101 gagccacatc cttactgcac tgagggagaa gcaggctcca gagctgagct tatctagtca
     2161 ggatttggag ttggttacca aggaagaccc caaagcactg gctgttgcct tgaactggga
     2221 cataaagaag acggagactg ttcaggaggc ctgtgagcgg gagctcgccc tgcgcctgca
     2281 gcagacgcag agcttgcatt ctctccggag catctcagca agcaaggcct caccacctgg
     2341 tgacctgcag aatcctaagc gagccagaca ggatcccaca tgactcgagt ctggtaaaga
     2401 aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg cagcttaatt
     2461 aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     2521 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     2581 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2641 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2701 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2761 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2821 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2881 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2941 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3001 aattttaaca aaatattaac gtttacaatt tctggcggca cgatggcatg agattatcaa
     3061 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3121 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3181 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3241 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3301 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3361 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3421 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3481 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3541 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     3601 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     3661 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     3721 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     3781 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     3841 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     3901 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     3961 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4021 aatcatgatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4081 atttagaaaa ataaacaaat aggtcatgac caaaatccct taacgtgagt tttcgttcca
     4141 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4201 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4261 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4321 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4381 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4441 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4501 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4561 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4621 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4681 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4741 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4801 ggccttttgc tggccttttg ctcacatgct ctttcctgcg ttatcccctg attctgtgga
     4861 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4921 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     4981 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     5041 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     5101 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5161 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5221 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     5281 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     5341 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     5401 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     5461 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     5521 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     5581 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     5641 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     5701 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     5761 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     5821 tcaacgacag gagcacgatc atgctagtca tgccccgcgc ccaccggaag gagctgactg
     5881 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     5941 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     6001 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     6061 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     6121 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     6181 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg
     6241 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     6301 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     6361 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     6421 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     6481 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     6541 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     6601 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     6661 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     6721 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     6781 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     6841 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     6901 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     6961 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     7021 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     7081 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     7141 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     7201 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     7261 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     7321 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     7381 cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat
//
