LOCUS       sequence-215251-        7758 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 7758)
  AUTHORS   DeLaBarre B, Christianson JC, Kopito RR, Brunger AT
  TITLE     Central pore residues mediate the p97/VCP activity required for 
            ERAD.
  JOURNAL   Mol Cell. 2006 May 19. 22(4):451-62.
  PUBMED    16713576
REFERENCE   2  (bases 1 to 7758)
  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: "Mol Cell. 
            2006 May 19. 22(4):451-62."
COMMENT     Sequence Label: pBD-0001
FEATURES             Location/Qualifiers
     source          1..7758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2636..2653)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2663..2680)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2699..2721
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2736..2760
                     /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)."
     primer_bind     complement(2760..2779)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2761..2779)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2863..2882)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3088..3165
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3166..4248
                     /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     complement(3185..3204)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5057..5248
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5242..5264)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5350..5489
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5405..5424
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5504..5521)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5675..6263)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5755..5774)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6374..6393)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6385..7200
                     /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             6385..7200
                     /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     complement(6458..6477)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(7292..7747)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7429..7450)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7641..7660
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tcccgtcact attagccata caggtcatcg tcattgtctt
      241 ctgtgtacac actgccacct gtgccacctc cactgccctg gctgggacca gctccaccct
      301 ggttccctga agggaatctg aagctgccaa aacctcgact ctgctgcagt gtctgggcga
      361 acatttcata cttccgaatg tcattatcgc tgacagaacg tcgggcaaaa cgcatggctt
      421 cctcaaagtg atctctgcgg atctcaggca ctggatcgtc ctcttctacc tccatagccg
      481 atggatttgt ctgcctctct cgttctcgcc taatctcact ctcaatagat tcacgaatgg
      541 ccagtttaca agcccgttgg caaatttctg tcaaatcagc tccagaaaag ccattagtca
      601 tcttagccag gaactccaaa tccacatcct tggcaactgg ggactttcgc agattggctt
      661 ttaggatggc aacacgggac ttctcatcag gaagtgggat ataaatgagc tgatctagac
      721 ggccaggtct taggatagca ggatcaatga tgtcaggcct gttggtagct ccaatgataa
      781 acacattctt ttttgtagac atgccatcca tttctgtcaa gatctgattg atgactcggt
      841 cagcagctcc accaccatct ccaatattac caccacgagc cttggcaatt gaatctaact
      901 catcaaagaa gagtacacag ggggcagctt gccgtgcctt gtcaaaaatt tcccggacat
      961 tggcctcaga ttccccaaac cacatggtaa gcagctcagg acccttgatg gagatgaagt
     1021 tggcctggca ttcattagca atggctttag ccagtaaggt tttcccacag ccaggaggtc
     1081 catagaaaag aacacctttg gagggagtca tgccaaattt gaggaatttg tctggatgtt
     1141 ccacggggta ctgaaccaac tcctgaagct cacgtttgac atcctccagg cctccaatat
     1201 cttcccaggt tacttgtggc acctctacca cagtttcccg aagtgctgat gggttgcttt
     1261 gactcaaagc ccaccggaag tcatccatag taactgccag ggaattcatg acctcagcat
     1321 caatggtctc atcttctagg tcaatgaggt ccattttttt ccggatggcc tgcagagcag
     1381 cctctgaaca tagggctgcc aaatcagcac caacatgacc atgagtctca ttggctacct
     1441 gttccaagtc cacatcatct gccagtttca tgttcttggt atggatctga agaatctcca
     1501 aacgtcctgt agcatcaggt attccaatat ctacctctct gtcaaagcga ccaaatcgcc
     1561 gtagggctgg gtcaatgctg ttgggtctat tggttgctgc cataactatc acatgtgctc
     1621 tctgctttag gccatccatg agggtcaaca actgagacac gatgcgacgc tccacttccc
     1681 catgagtttt ctctcttttg ggtgcaatgg catcaagctc atcgatgaag atgatagcag
     1741 gagcattctt ttcagcttcc tcaaaggctt tacgaaggtt gctctcagac tcaccagcca
     1801 atttgctcat gatttcagga ccattgatca gaaagaagaa ggctccagtt tcatttgcca
     1861 cagctcgagc aatcagggtc ttccctgtcc caggaggccc atacaacaag attccccgag
     1921 gaggctttac accaatcgcc ttgaagagcg caggatgtct cagtggcagc tccaccatct
     1981 cctttatctg agctagctgc ttcctgcaac caccgacgtc atcatagcct acttcattca
     2041 aggattcctc ctcatcctct cgtttgattg gctccccctc acagtggatc actgtgtctg
     2101 gagcaacaat acagtaaggg ctgggatctg tctctacaac tttgaactcc acagcacgca
     2161 tcccaccccg gacaagaaaa atatctcctt tacggatggg ccgataagct tccaggaagt
     2221 acggcttaag gtatacctca aagagattgc cagtgatgcc ttccactgtg tcatcgatgg
     2281 gtagaacgtg gatacgtttg ccatacttta catcagggca tggctggatg ctgatgacat
     2341 ctcctaggcg aactcggagg ttattccgaa caactctatt cattcgaatc ttctcatcag
     2401 aacacgtgtc atcagaaaga acaatgcata cagcttcccg tcttttcttt ccttttagca
     2461 acaccgtgtc acctcggaac aactgcagtt catccatctt gggctgggac aaggacacca
     2521 cgctgttatc ttcattgatg gcttcatcaa caattaaccg attgggtcgg ttcttctgtt
     2581 tgagaatggc tgttgataaa tcatcacctt ttgaatcggc tccagaggcc atatggctgc
     2641 cgcgcggcac caggccgctg ctgtgatgat gatgatgatg gctgctgccc atggtatatc
     2701 tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc tcacaattcc
     2761 cctatagtga gtcgtattaa tttcgcggga tcgagatctc gatcctctac gccggacgca
     2821 tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca
     2881 ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta
     2941 tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc
     3001 ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt
     3061 cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt
     3121 atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg
     3181 ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac
     3241 caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg
     3301 aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc
     3361 gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct
     3421 cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa
     3481 gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac
     3541 tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg
     3601 ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac
     3661 ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta
     3721 gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc
     3781 actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt
     3841 tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc
     3901 aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt
     3961 gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg
     4021 ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg
     4081 caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa
     4141 agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     4201 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     4261 taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt
     4321 caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac
     4381 tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg
     4441 cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat
     4501 cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa
     4561 gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt
     4621 gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg
     4681 cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat
     4741 ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat
     4801 tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt
     4861 attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac
     4921 cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg
     4981 tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc
     5041 ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat
     5101 cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc
     5161 agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg
     5221 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     5281 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     5341 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac
     5401 tatgcggcat cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca
     5461 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     5521 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5581 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5641 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5701 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5761 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5821 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5881 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5941 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6001 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6061 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     6121 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6181 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6241 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6301 agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact gtctgcttac
     6361 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctctagg
     6421 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat
     6481 gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg
     6541 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
     6601 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat
     6661 gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa
     6721 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat
     6781 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg
     6841 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
     6901 tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca
     6961 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
     7021 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     7081 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt
     7141 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
     7201 gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7261 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
     7321 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     7381 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     7441 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     7501 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     7561 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     7621 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     7681 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     7741 ggcgcgtccc attcgcca
//
