LOCUS       sequence-171186-        6160 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 6160)
  AUTHORS   Beresford PJ, Zhang D, Oh DY, Fan Z, Greer EL, Russo ML, Jaju M, 
            Lieberman J
  TITLE     Granzyme A activates an endoplasmic reticulum-associated 
            caspase-independent nuclease to induce single-stranded DNA nicks.
  JOURNAL   J Biol Chem 2001 Nov 16;276(46):43285-93. Epub 2001 Sep 12.
  PUBMED    11555662
REFERENCE   2  (bases 1 to 6160)
  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"; date: "2001-11-16"; volume: "276(46)"; pages: "43285-93. Epub
            2001 Sep 12"
COMMENT     Sequence Label: pET30a-PP32
FEATURES             Location/Qualifiers
     source          1..6160
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     868..887
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(907..984)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1190..1209
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1297..1316
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1297..1315
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1316..1340
                     /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             1355..1377
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1388..1405
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1415..1432
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1439..1483
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1499..1513
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             2317..2334
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2387..2405)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2401..2448
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2485..2940
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2572..2591)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2782..2803
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3032..3847)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3032..3847)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3755..3774
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3839..3858
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3969..4557
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4458..4477
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4711..4728
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4743..4882
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4808..4827)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4968..4990
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4984..5175)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5984..6160,1..906))
                     /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"
ORIGIN
        1 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
       61 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg
      121 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
      181 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
      241 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
      301 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
      361 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
      421 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
      481 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
      541 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
      601 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
      661 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
      721 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
      781 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
      841 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
      901 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
      961 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     1021 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     1081 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     1141 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     1201 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     1261 cgtagaggat cgagatcgat ctcgatcccg cgaaattaat acgactcact ataggggaat
     1321 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     1381 acatatgcac catcatcatc atcattcttc tggtctggtg ccacgcggtt ctggtatgaa
     1441 agaaaccgct gctgctaaat tcgaacgcca gcacatggac agcccagatc tgggtaccga
     1501 cgacgacgac aaggccatgg agatgggcag acggattcat ttagagctgc ggaacaggac
     1561 gccctctgat gtgaaagaac ttgtcctgga caacagtcgg tcgaatgaag gcaaactcga
     1621 aggcctcaca gatgaatttg aagaactgga attcttaagt acaatcaacg taggcctcac
     1681 ctcaatcgca aacttaccaa agttaaacaa acttaagaag cttgaactaa gcgataacag
     1741 agtctcaggg ggcctggaag tattggcaga aaagtgtccg aacctcacgc atctaaattt
     1801 aagtggcaac aaaattaaag acctcagcac aatagagcca ctgaaaaagt tagaaaacct
     1861 caagagctta gaccttttca attgcgaggt aaccaacctg aacgactacc gagaaaatgt
     1921 gttcaagctc ctcccgcaac tcacatatct cgacggctat gaccgggacg acaaggaggc
     1981 ccctgactcg gatgctgagg gctacgtgga gggcctggat gatgaggagg aggatgagga
     2041 tgaggaggag tatgatgaag atgctcaggt agtggaagac gaggaggacg aggatgagga
     2101 ggaggaaggt gaagaggagg acgtgagtgg agaggaggag gatgaagaag gttataacga
     2161 tggagaggta gatgacgagg aagatgaaga agagcttggt gaagaagaaa ggggtcagaa
     2221 gcgaaaacga gaacctgaag atgagggaca agatgatgac taagtggagg ggatccgaat
     2281 tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
     2341 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     2401 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2461 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2521 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2581 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2641 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2701 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2761 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2821 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2881 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2941 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3001 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     3061 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     3121 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     3181 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     3241 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     3301 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     3361 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     3421 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3481 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     3541 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3601 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     3661 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3721 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3781 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     3841 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     3901 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3961 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4021 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4081 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4141 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4201 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4261 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4321 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4381 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4441 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4501 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4561 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4621 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4681 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4741 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4801 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4861 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4921 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4981 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     5041 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     5101 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     5161 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     5221 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     5281 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     5341 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     5401 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     5461 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     5521 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     5581 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     5641 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     5701 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     5761 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     5821 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     5881 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5941 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     6001 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6061 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     6121 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
//
