LOCUS       sequence-175052-        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 - as 
            control - 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 - controls
  JOURNAL   Unpublished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            "Unpublished"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-Arg-e-Delta-K-Flv
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     75..98
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    171..195
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             342..431
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(883..907)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      975..1069
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1113..1772
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1179..1198)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1837..1923
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2016..3098)
                     /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     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /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(3260..3282)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3368..3508
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3423..3442
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3523..3540)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3694..4282)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3774..3793)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4453..5244,5245..5313))
                     /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     5076..5095
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5314..5418)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5486..5504
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5528..5542,5543..5548,5549..5565,5566..5572)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    5548..5564
                     /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    5580..5596
                     /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             5615..5626
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             5639..5662
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             join(5666..6662,1..101)
                     /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 tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg
       61 gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac taattcgagc tcgaacaaca
      121 acaacaataa caataacaac aacctcggga tcgacgaaaa cctgtacctc acaagtttgt
      181 acaaaaaagc aggcttagag aatctttatt ttcagagaca cggatctgga gcttggctgt
      241 tgcccgtctc actggtgaga agaagaacca ccctggcgcc caatacgcaa accgcctctc
      301 cccgcgcgtt ggccgattca ttaatgcagg gaatgggatc ctacccatac gatgttcctg
      361 actatgcggg ctatccctat gacgtcccgg actatgcagg atcctatcca tacgacgttc
      421 cagattacgc tggtgctgca gatatcactc ttatcagcgg cagcaccctc ggcggtgccg
      481 aatatgtagc agaacacctg gctgaaaagc tggaagaggc gggttttacc accgaaacgc
      541 tgcacggtcc gctgttggaa gatttatctg cctcggggat ctggctgatt atcagctcca
      601 cccacggtgc cggagatatt ccggacaacc tttctccttt ctatgaagca ttgcaggaac
      661 agaagcccga tctttctgcg gtccgctttg gcgcaatcgg tattggtagt cgtgaatatg
      721 acaccttttg tggggctatc gataaactcg aggcagaact caaagattcc ggtgcaaaac
      781 agacaggcga aacactgaag atcaacattc ttgatcacga cattccggaa gatccggcag
      841 aagaatggct gggatcgtgg attaatttac tcaaataatg atacccagct ttcttgtaca
      901 aagtggtgat ggcgcgcctg taggacgtcg acggtaccat cgatacgcgt tcgaagctta
      961 attagctgag cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga
     1021 tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc caagctagct
     1081 tggcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca
     1141 ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc
     1201 aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga
     1261 aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc
     1321 atccggaatt tcgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc
     1381 cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc
     1441 acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa
     1501 acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct
     1561 gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg
     1621 ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc
     1681 aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac
     1741 agtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat tggtgccctt
     1801 aaacgcctgg ggtaatgact ctctagcttg aggcatcaaa taaaacgaaa ggctcagtcg
     1861 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     1921 aatccgccct ctagattacg tgcagtcgat gataagctgt caaacatgag aattgtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc accattcgat ggtgtcggaa
     3181 tttcgggcag cgttgggtcc tggccacggg tgcgcatgat ctagagctgc ctcgcgcgtt
     3241 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     3301 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     3361 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     3421 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     3481 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     3541 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3601 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3661 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3721 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3781 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3841 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3901 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3961 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4021 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4081 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     4141 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4201 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4261 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4321 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4381 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4441 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4501 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4561 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4621 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4681 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4741 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4801 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4861 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4921 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4981 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5041 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5101 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5161 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5221 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5281 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     5341 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5401 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     5461 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcacct
     5521 cgagaaatca taaaaaattt atttgctttg tgagcggata acaattataa tagattcaat
     5581 tgtgagcgga taacaatttc acacagaatt cattaaagag gagaaattaa ccatgggaca
     5641 tcaccatcat caccatcacc attcaaaaat cgaagaaggt aaactggtaa tctggattaa
     5701 cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga aagataccgg
     5761 aattaaagtc accgttgagc atccggataa actggaagag aaattcccac aggttgcggc
     5821 aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg gctacgctca
     5881 atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc tgtatccgtt
     5941 tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg ctgttgaagc
     6001 gttatcgctg atttataaca aagatctgct gccgaacccg ccaaaaacct gggaagagat
     6061 cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca
     6121 agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt tcaagtatga
     6181 aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcga aagcgggtct
     6241 gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg attactccat
     6301 cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg
     6361 gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga ccttcaaggg
     6421 tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa
     6481 caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag gtctggaagc
     6541 ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg aagagttggc
     6601 gaaagatcca cgtattgccg ccactatgga aaacgcccag aaaggtgaaa tcatgccgaa
     6661 ca
//
