LOCUS       sequence-173970-        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  JOURNAL   Unpublished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            "Unpublished"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-Gly-eK-Flv
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     914..933
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(953..1030)
                     /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(1114..1136)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1222..1362
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1277..1296
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1377..1394)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1548..2136)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1628..1647)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2307..3098,3099..3167))
                     /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     2930..2949
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3168..3272)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3340..3358
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3382..3396,3397..3402,3403..3419,3420..3426)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3402..3418
                     /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    3434..3450
                     /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             3469..3480
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3493..3516
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3520..4617
                     /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"
     primer_bind     4591..4614
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    4687..4711
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4714..4734
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4858..4947
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(5399..5423)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5491..5585
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5629..6288
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5695..5714)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6353..6439
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6532..6662,1..952))
                     /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 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
       61 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
      121 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
      181 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
      241 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
      301 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
      361 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
      421 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
      481 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
      541 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
      601 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
      661 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
      721 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
      781 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
      841 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
      901 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
      961 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt
     1021 cgatggtgtc ggaatttcgg gcagcgttgg gtcctggcca cgggtgcgca tgatctagag
     1081 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     1141 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     1201 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     1261 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     1321 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg
     1381 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     1441 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     1501 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     1561 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     1621 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     1681 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1741 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1801 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1861 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1921 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     1981 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2041 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2101 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2161 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     2221 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2281 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2341 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2401 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     2461 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     2521 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     2581 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     2641 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     2701 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     2761 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     2821 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     2881 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     2941 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3001 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3061 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3121 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3181 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3241 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     3301 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     3361 tttcgtcttc acctcgagaa atcataaaaa atttatttgc tttgtgagcg gataacaatt
     3421 ataatagatt caattgtgag cggataacaa tttcacacag aattcattaa agaggagaaa
     3481 ttaaccatgg gacatcacca tcatcaccat caccattcaa aaatcgaaga aggtaaactg
     3541 gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc
     3601 gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc
     3661 ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt
     3721 ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac
     3781 aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg
     3841 atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa
     3901 acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg
     3961 atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat
     4021 gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc
     4081 gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac
     4141 accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac
     4201 ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg
     4261 ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac
     4321 gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct gctgactgat
     4381 gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac
     4441 gaggaagagt tggcgaaaga tccacgtatt gccgccacta tggaaaacgc ccagaaaggt
     4501 gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg
     4561 atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagactaat
     4621 tcgagctcga acaacaacaa caataacaat aacaacaacc tcgggatcga cgaaaacctg
     4681 tacctcacaa gtttgtacaa aaaagcaggc ttagagaatc tttattttca gggtcacgga
     4741 tctggagctt ggctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     4801 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagggaat gggatcctac
     4861 ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggatcc
     4921 tatccatacg acgttccaga ttacgctggt gctgcagata tcactcttat cagcggcagc
     4981 accctcggcg gtgccgaata tgtagcagaa cacctggctg aaaagctgga agaggcgggt
     5041 tttaccaccg aaacgctgca ctgtccgctg ttggaagatt tatctgcctc ggggatctgg
     5101 ctgattatca gctccaccca cggtgccgga gatattccgg acaacctttc tcctttctat
     5161 gaagcattgc aggaacagaa gcccgatctt tctgcggtcc gctttggcgc aatcggtatt
     5221 ggtagtcgtg aatatgacac cttttgtggg gctatcgata aactcgaggc agaactcaaa
     5281 gattccggtg caaaacagac aggcgaaaca ctgaagatca acattcttga tcacgacatt
     5341 ccggaagatc cggcagaaga atggctggga tcgtggatta atttactcaa ataatgatac
     5401 ccagctttct tgtacaaagt ggtgatggcg cgcctgtagg acgtcgacgg taccatcgat
     5461 acgcgttcga agcttaatta gctgagcttg gactcctgtt gatagatcca gtaatgacct
     5521 cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg
     5581 agaatccaag ctagcttggc gagattttca ggagctaagg aagctaaaat ggagaaaaaa
     5641 atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca
     5701 tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt
     5761 ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc
     5821 cgcctgatga atgctcatcc ggaatttcgt atggcaatga aagacggtga gctggtgata
     5881 tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg
     5941 ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg
     6001 gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc
     6061 gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac
     6121 aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga caaggtgctg
     6181 atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg
     6241 cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaatt tttttaaggc
     6301 agttattggt gcccttaaac gcctggggta atgactctct agcttgaggc atcaaataaa
     6361 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     6421 tctcctgagt aggacaaatc cgccctctag attacgtgca gtcgatgata agctgtcaaa
     6481 catgagaatt gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     6541 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     6601 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     6661 ca
//
