LOCUS       sequence-175068-        6662 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 6662)
  TITLE     artificial N-end rule substrates
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6662)
  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            "Unpublished"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-Asp-eK-Flv
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     140..163
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    236..260
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             407..496
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(948..972)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      1040..1134
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1178..1837
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1244..1263)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1902..1988
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2081..3163)
                     /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     3125..3144
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3164..3241)
                     /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(3325..3347)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3433..3573
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3488..3507
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3588..3605)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3759..4347)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3839..3858)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4518..5309,5310..5378))
                     /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     5141..5160
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5379..5483)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5551..5569
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5593..5607,5608..5613,5614..5630,5631..5637)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    5613..5629
                     /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)."
     protein_bind    5645..5661
                     /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             5680..5691
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             5704..5727
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             join(5731..6662,1..166)
                     /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 ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc cagaaaggtg aaatcatgcc
       61 gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga tcaacgccgc
      121 cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagactaatt cgagctcgaa
      181 caacaacaac aataacaata acaacaacct cgggatcgac gaaaacctgt acctcacaag
      241 tttgtacaaa aaagcaggct tagaaaacct gtattttcag gatcacggat ctggagcttg
      301 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
      361 ctctccccgc gcgttggccg attcattaat gcagggaatg ggatcctacc catacgatgt
      421 tcctgactat gcgggctatc cctatgacgt cccggactat gcaggatcct atccatacga
      481 cgttccagat tacgctggtg ctgcagatat cactcttatc agcggcagca ccctcggcgg
      541 tgccgaatat gtagcagaac acctggctga aaagctggaa gaggcgggtt ttaccaccga
      601 aacgctgcac ggtccgctgt tggaagattt atctgcctcg gggatctggc tgattatcag
      661 ctccacccac ggtgccggag atattccgga caacctttct cctttctatg aagcattgca
      721 ggaacagaag cccgatcttt ctgcggtccg ctttggcgca atcggtattg gtagtcgtga
      781 atatgacacc ttttgtgggg ctatcgataa actcgaggca gaactcaaag attccggtgc
      841 aaaacagaca ggcgaaacac tgaagatcaa cattcttgat cacgacattc cggaagatcc
      901 ggcagaagaa tggctgggat cgtggattaa tttactcaaa taatgatacc cagctttctt
      961 gtacaaagtg gtgatggcgc gcctgtagga cgtcgacggt accatcgata cgcgttcgaa
     1021 gcttaattag ctgagcttgg actcctgttg atagatccag taatgacctc agaactccat
     1081 ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaagc
     1141 tagcttggcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
     1201 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
     1261 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
     1321 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
     1381 tgctcatccg gaatttcgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
     1441 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
     1501 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
     1561 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
     1621 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
     1681 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
     1741 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
     1801 acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca gttattggtg
     1861 cccttaaacg cctggggtaa tgactctcta gcttgaggca tcaaataaaa cgaaaggctc
     1921 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta
     1981 ggacaaatcc gccctctaga ttacgtgcag tcgatgataa gctgtcaaac atgagaattg
     2041 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     2101 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     2161 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     2221 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     2281 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     2341 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     2401 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     2461 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     2521 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     2581 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     2641 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     2701 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     2761 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     2821 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     2881 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     2941 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     3001 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     3061 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     3121 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     3181 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcaccat tcgatggtgt
     3241 cggaatttcg ggcagcgttg ggtcctggcc acgggtgcgc atgatctaga gctgcctcgc
     3301 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     3361 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     3421 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     3481 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     3541 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     3601 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3661 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3721 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3781 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3841 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3901 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3961 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4021 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4081 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4141 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     4201 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4261 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4321 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4381 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4441 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4501 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4561 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4621 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4681 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4741 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4801 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4861 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4921 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4981 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5041 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5101 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5161 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5221 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5281 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5341 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5401 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5461 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     5521 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt
     5581 cacctcgaga aatcataaaa aatttatttg ctttgtgagc ggataacaat tataatagat
     5641 tcaattgtga gcggataaca atttcacaca gaattcatta aagaggagaa attaaccatg
     5701 ggacatcacc atcatcacca tcaccattca aaaatcgaag aaggtaaact ggtaatctgg
     5761 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
     5821 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
     5881 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
     5941 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
     6001 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
     6061 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
     6121 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
     6181 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
     6241 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
     6301 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
     6361 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     6421 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     6481 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     6541 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     6601 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     6661 tt
//
