LOCUS       sequence-251066-        6424 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6424)
  AUTHORS   Muzio M, Salvesen GS, Dixit VM
  TITLE     FLICE induced apoptosis in a cell-free system. Cleavage of caspase 
            zymogens.
  JOURNAL   J Biol Chem. 1997 Jan 31. 272(5):2952-6.
  PUBMED    9006941
REFERENCE   2  (bases 1 to 6424)
  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"; journalName: "J Biol 
            Chem. 1997 Jan 31. 272(5):2952-6."
COMMENT     Sequence Label: pcDNA3-CrmA
FEATURES             Location/Qualifiers
     source          1..6424
                     /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             678..701
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(1752..1770)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1753..1770)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1790..1807)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1796..2020
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2066..2494
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2153..2172)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2363..2384
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2503..2523)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2508..2837
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2688..2823
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2750..2769
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2904..3698
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2958..2977)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3568..3587
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3872..3993
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3909..3928)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3963..3982
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4042..4058)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4042..4058)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4055..4077)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4066..4082
                     /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)."
     promoter        complement(join(4090..4096,4097..4114,4115..4120))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4135..4156
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4273..4290)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4444..5032)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4524..4543)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5203..5994,5995..6063))
                     /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     5826..5845
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6064..6168)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6243..6262)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atacgcgttg 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 cccaagcttc catggcagac tacaaggacg acgatgacaa ggatatcttc agggaaatcg
      721 catcttctat gaaaggagag aatgtattca tttctccacc gtcaatctcg tcagtattga
      781 caatactgta ttatggagct aatggatcca ctgctgaaca gctatcaaaa tatgtagaaa
      841 aggaggcgga caagaataag gatgatatct cattcaagtc catgaataaa gtatatgggc
      901 gatattctgc agtgtttaaa gattcctttt tgagaaaaat tggagataat ttccaaactg
      961 ttgacttcac tgattgtcgc actgtagatg cgatcaacaa gtgtgttgat atcttcactg
     1021 aggggaaaat taatccacta ttggatgaac cattgtctcc agatacctgt ctcctagcaa
     1081 ttagtgccgt atactttaaa gcaaaatggt tgatgccatt tgaaaaggaa tttaccagtg
     1141 attatccctt ttacgtatct ccaacggaaa tggtagatgt aagtatgatg tctatgtacg
     1201 gcgaggcatt taatcacgca tctgtaaaag aatcattcgg caacttttca atcatagaac
     1261 tgccatatgt tggagatact agtatggtgg taattcttcc agacaatatt gatggactag
     1321 aatccataga acaaaatcta acagatacaa attttaagaa atggtgtgac tctatggatg
     1381 ctatgtttat cgatgtgcac attcccaagt ttaaggtaac aggctcgtat aatctggtgg
     1441 atgcgctagt aaagttggga ctgacagagg tgttcggttc aactggagat tatagcaata
     1501 tgtgtaattc agatgtgagt gtcgacgcta tgatccacaa aacgtatata gatgtcaatg
     1561 aagagtatac agaagcagct gcagcaactt gtgcgctggt ggcagactgt gcatcaacag
     1621 ttacaaatga gttctgtgca gatcatccgt tcatctatgt gattaggcat gtcgatggca
     1681 aaattctttt cgttggtaga tattgctctc caacaactaa ttaactcgag catgcatcta
     1741 gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc ctcgactgtg
     1801 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1861 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1921 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     1981 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2041 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2101 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2161 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2221 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2281 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2341 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2401 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2461 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     2521 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2581 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2641 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2701 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2761 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2821 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     2881 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     2941 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3001 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3061 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     3121 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3181 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3241 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3301 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3361 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3421 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3481 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3541 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3601 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3661 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     3721 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     3781 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3841 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     3901 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3961 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4021 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4081 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4141 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4201 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4261 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4321 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4381 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4441 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4501 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4561 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4621 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4681 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4741 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4801 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4861 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4921 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4981 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5041 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5101 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5161 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5221 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5281 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5341 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5401 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5461 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5521 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5581 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5641 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5701 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5761 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5821 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5881 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5941 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6001 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6061 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6121 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6181 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     6241 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     6301 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     6361 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     6421 agat
//
