LOCUS       sequence-173975-        6630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression vector pVP16 containing a derivative of E. coli
            lacZ fused with E. coli flavin binding protein MioC to be used in 
            N-end rule in vitro ubiquitination studies.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6630)
  TITLE     artificial N-end rule substrates
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6630)
  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: 
            "Unpublished"
COMMENT     Sequence Label: His8:MBP-tev-Arg-eK-Flv
FEATURES             Location/Qualifiers
     source          1..6630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     76..99
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    172..196
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             343..432
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(884..908)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      976..1070
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1114..1773
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1180..1199)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1838..1924
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2017..3099)
                     /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     3061..3080
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3100..3177)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(3261..3283)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3369..3509
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3424..3443
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3524..3541)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3695..4283)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3775..3794)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4454..5245,5246..5314))
                     /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     5077..5096
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5315..5419)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5487..5505
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     protein_bind    5549..5565
                     /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             5584..5595
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             5608..5631
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             join(5635..6630,1..102)
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
ORIGIN
        1 atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc
       61 ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag ctcgaacaac
      121 aacaacaata acaataacaa caacctcggg atcgacgaaa acctgtacct cacaagtttg
      181 tacaaaaaag caggcttaga gaatctttat tttcagagac acggatctgg agcttggctg
      241 ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct
      301 ccccgcgcgt tggccgattc attaatgcag ggaatgggat cctacccata cgatgttcct
      361 gactatgcgg gctatcccta tgacgtcccg gactatgcag gatcctatcc atacgacgtt
      421 ccagattacg ctggtgctgc agatatcact cttatcagcg gcagcaccct cggcggtgcc
      481 gaatatgtag cagaacacct ggctgaaaag ctggaagagg cgggttttac caccgaaacg
      541 ctgcacggtc cgctgttgga agatttatct gcctcgggga tctggctgat tatcagctcc
      601 acccacggtg ccggagatat tccggacaac ctttctcctt tctatgaagc attgcaggaa
      661 cagaagcccg atctttctgc ggtccgcttt ggcgcaatcg gtattggtag tcgtgaatat
      721 gacacctttt gtggggctat cgataaactc gaggcagaac tcaaagattc cggtgcaaaa
      781 cagacaggcg aaacactgaa gatcaacatt cttgatcacg acattccgga agatccggca
      841 gaagaatggc tgggatcgtg gattaattta ctcaaataat gatacccagc tttcttgtac
      901 aaagtggtga tggcgcgcct gtaggacgtc gacggtacca tcgatacgcg ttcgaagctt
      961 aattagctga gcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg
     1021 atttgttcag aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaagctagc
     1081 ttggcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc
     1141 accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct
     1201 caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag
     1261 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct
     1321 catccggaat ttcgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac
     1381 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac
     1441 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa
     1501 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc
     1561 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc
     1621 gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt
     1681 caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa
     1741 cagtactgcg atgagtggca gggcggggcg taattttttt aaggcagtta ttggtgccct
     1801 taaacgcctg gggtaatgac tctctagctt gaggcatcaa ataaaacgaa aggctcagtc
     1861 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     1921 aaatccgccc tctagattac gtgcagtcga tgataagctg tcaaacatga gaattgtgcc
     1981 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2041 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2101 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     2161 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     2221 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     2281 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     2341 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     2401 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     2461 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     2521 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     2581 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     2641 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     2701 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     2761 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     2821 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     2881 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     2941 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     3001 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     3061 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     3121 ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcga tggtgtcgga
     3181 atttcgggca gcgttgggtc ctggccacgg gtgcgcatga tctagagctg cctcgcgcgt
     3241 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     3301 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     3361 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact
     3421 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     3481 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     3541 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3601 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3661 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3721 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3781 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3841 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3901 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3961 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4021 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4081 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     4141 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4201 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4261 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4321 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4381 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4441 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4501 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4561 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4621 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4681 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4741 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4801 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4861 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4921 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4981 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5041 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5101 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5161 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5221 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5281 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5341 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5401 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     5461 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcacc
     5521 tcgagaaatc ataaaaaatt tatttgcttt gtgagcggat aacaatttca cacagaattc
     5581 attaaagagg agaaattaac catgggacat caccatcatc accatcacca ttcaaaaatc
     5641 gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa
     5701 gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa
     5761 ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg
     5821 gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac
     5881 aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag
     5941 ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg
     6001 ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa
     6061 ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct
     6121 gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc
     6181 gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa
     6241 cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca
     6301 gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat
     6361 ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg
     6421 agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac
     6481 tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta
     6541 gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa
     6601 aacgcccaga aaggtgaaat catgccgaac
//
