LOCUS       sequence-175064-        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  JOURNAL   Unpublished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            "Unpublished"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-Lys-eK-Flv
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     564..583
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(603..680)
                     /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(764..786)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    872..1012
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     927..946
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1027..1044)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1198..1786)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1278..1297)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1957..2748,2749..2817))
                     /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     2580..2599
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2818..2922)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2990..3008
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3032..3046,3047..3052,3053..3069,3070..3076)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3052..3068
                     /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    3084..3100
                     /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             3119..3130
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3143..3166
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3170..4267
                     /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     4241..4264
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    4337..4361
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4508..4597
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(5049..5073)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5141..5235
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5279..5938
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5345..5364)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6003..6089
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6182..6662,1..602))
                     /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 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
       61 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
      121 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
      181 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
      241 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
      301 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
      361 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
      421 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
      481 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
      541 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
      601 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
      661 ttgcaccatt cgatggtgtc ggaatttcgg gcagcgttgg gtcctggcca cgggtgcgca
      721 tgatctagag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
      781 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
      841 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
      901 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
      961 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
     1021 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     1081 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     1141 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     1201 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     1261 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     1321 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     1381 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     1441 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     1501 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     1561 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     1621 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     1681 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     1741 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     1801 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     1861 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     1921 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     1981 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     2041 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     2101 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     2161 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     2221 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     2281 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     2341 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     2401 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     2461 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     2521 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     2581 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     2641 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     2701 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     2761 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     2821 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     2881 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     2941 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     3001 cacgaggccc tttcgtcttc acctcgagaa atcataaaaa atttatttgc tttgtgagcg
     3061 gataacaatt ataatagatt caattgtgag cggataacaa tttcacacag aattcattaa
     3121 agaggagaaa ttaaccatgg gacatcacca tcatcaccat caccattcaa aaatcgaaga
     3181 aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg
     3241 taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga
     3301 agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca
     3361 cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc
     3421 gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat
     3481 tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa
     3541 cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa
     3601 gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga
     3661 cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga
     3721 taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat
     3781 gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat
     3841 gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt
     3901 aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc
     3961 aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct
     4021 gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct
     4081 gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacta tggaaaacgc
     4141 ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg
     4201 tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc
     4261 gcagactaat tcgagctcga acaacaacaa caataacaat aacaacaacc tcgggatcga
     4321 cgaaaacctg tacctcacaa gtttgtacaa aaaagcaggc ttagaaaacc tgtattttca
     4381 gaaacacgga tctggagctt ggctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     4441 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagggaat
     4501 gggatcctac ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta
     4561 tgcaggatcc tatccatacg acgttccaga ttacgctggt gctgcagata tcactcttat
     4621 cagcggcagc accctcggcg gtgccgaata tgtagcagaa cacctggctg aaaagctgga
     4681 agaggcgggt tttaccaccg aaacgctgca cggtccgctg ttggaagatt tatctgcctc
     4741 ggggatctgg ctgattatca gctccaccca cggtgccgga gatattccgg acaacctttc
     4801 tcctttctat gaagcattgc aggaacagaa gcccgatctt tctgcggtcc gctttggcgc
     4861 aatcggtatt ggtagtcgtg aatatgacac cttttgtggg gctatcgata aactcgaggc
     4921 agaactcaaa gattccggtg caaaacagac aggcgaaaca ctgaagatca acattcttga
     4981 tcacgacatt ccggaagatc cggcagaaga atggctggga tcgtggatta atttactcaa
     5041 ataatgatac ccagctttct tgtacaaagt ggtgatggcg cgcctgtagg acgtcgacgg
     5101 taccatcgat acgcgttcga agcttaatta gctgagcttg gactcctgtt gatagatcca
     5161 gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt
     5221 tttattggtg agaatccaag ctagcttggc gagattttca ggagctaagg aagctaaaat
     5281 ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca
     5341 ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat
     5401 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
     5461 cattcttgcc cgcctgatga atgctcatcc ggaatttcgt atggcaatga aagacggtga
     5521 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac
     5581 gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc
     5641 gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa
     5701 tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc
     5761 caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga
     5821 caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt
     5881 cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaatt
     5941 tttttaaggc agttattggt gcccttaaac gcctggggta atgactctct agcttgaggc
     6001 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     6061 cggtgaacgc tctcctgagt aggacaaatc cgccctctag attacgtgca gtcgatgata
     6121 agctgtcaaa catgagaatt gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     6181 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     6241 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     6301 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     6361 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     6421 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     6481 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     6541 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     6601 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     6661 gc
//
