LOCUS       sequence-175037-        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  JOURNAL   Unpublished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            "Unpublished"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-Phe-e-Delta-K-Flv
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     974..993
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1013..1090)
                     /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(1174..1196)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1282..1422
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1337..1356
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1437..1454)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1608..2196)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1688..1707)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2367..3158,3159..3227))
                     /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     2990..3009
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3228..3332)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3400..3418
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3442..3456,3457..3462,3463..3479,3480..3486)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3462..3478
                     /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    3494..3510
                     /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             3529..3540
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3553..3576
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3580..4677
                     /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     4651..4674
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    4747..4771
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4918..5007
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(5459..5483)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5551..5645
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5689..6348
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5755..5774)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6413..6499
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6592..6662,1..1012))
                     /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 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
       61 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
      121 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
      181 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
      241 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
      301 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
      361 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
      421 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
      481 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
      541 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
      601 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
      661 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
      721 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
      781 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
      841 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
      901 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
      961 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     1021 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt
     1081 cgatggtgtc ggaatttcgg gcagcgttgg gtcctggcca cgggtgcgca tgatctagag
     1141 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     1201 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     1261 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     1321 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     1381 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg
     1441 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     1501 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     1561 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     1621 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     1681 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     1741 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1801 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1861 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1921 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1981 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2041 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2101 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2161 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2221 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     2281 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2341 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2401 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2461 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     2521 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     2581 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     2641 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     2701 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     2761 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     2821 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     2881 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     2941 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3001 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3061 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3121 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3181 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3241 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3301 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     3361 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     3421 tttcgtcttc acctcgagaa atcataaaaa atttatttgc tttgtgagcg gataacaatt
     3481 ataatagatt caattgtgag cggataacaa tttcacacag aattcattaa agaggagaaa
     3541 ttaaccatgg gacatcacca tcatcaccat caccattcaa aaatcgaaga aggtaaactg
     3601 gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc
     3661 gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc
     3721 ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt
     3781 ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac
     3841 aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg
     3901 atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa
     3961 acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg
     4021 atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat
     4081 gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc
     4141 gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac
     4201 accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac
     4261 ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg
     4321 ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac
     4381 gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct gctgactgat
     4441 gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac
     4501 gaggaagagt tggcgaaaga tccacgtatt gccgccacta tggaaaacgc ccagaaaggt
     4561 gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg
     4621 atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagactaat
     4681 tcgagctcga acaacaacaa caataacaat aacaacaacc tcgggatcga cgaaaacctg
     4741 tacctcacaa gtttgtacaa aaaagcaggc ttagagaatc tttattttca gttccacgga
     4801 tctggagctt ggctgttgcc cgtctcactg gtgagaagaa gaaccaccct ggcgcccaat
     4861 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagggaat gggatcctac
     4921 ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggatcc
     4981 tatccatacg acgttccaga ttacgctggt gctgcagata tcactcttat cagcggcagc
     5041 accctcggcg gtgccgaata tgtagcagaa cacctggctg aaaagctgga agaggcgggt
     5101 tttaccaccg aaacgctgca cggtccgctg ttggaagatt tatctgcctc ggggatctgg
     5161 ctgattatca gctccaccca cggtgccgga gatattccgg acaacctttc tcctttctat
     5221 gaagcattgc aggaacagaa gcccgatctt tctgcggtcc gctttggcgc aatcggtatt
     5281 ggtagtcgtg aatatgacac cttttgtggg gctatcgata aactcgaggc agaactcaaa
     5341 gattccggtg caaaacagac aggcgaaaca ctgaagatca acattcttga tcacgacatt
     5401 ccggaagatc cggcagaaga atggctggga tcgtggatta atttactcaa ataatgatac
     5461 ccagctttct tgtacaaagt ggtgatggcg cgcctgtagg acgtcgacgg taccatcgat
     5521 acgcgttcga agcttaatta gctgagcttg gactcctgtt gatagatcca gtaatgacct
     5581 cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg
     5641 agaatccaag ctagcttggc gagattttca ggagctaagg aagctaaaat ggagaaaaaa
     5701 atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca
     5761 tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt
     5821 ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc
     5881 cgcctgatga atgctcatcc ggaatttcgt atggcaatga aagacggtga gctggtgata
     5941 tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg
     6001 ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg
     6061 gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc
     6121 gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac
     6181 aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga caaggtgctg
     6241 atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg
     6301 cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaatt tttttaaggc
     6361 agttattggt gcccttaaac gcctggggta atgactctct agcttgaggc atcaaataaa
     6421 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     6481 tctcctgagt aggacaaatc cgccctctag attacgtgca gtcgatgata agctgtcaaa
     6541 catgagaatt gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     6601 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     6661 ag
//
