LOCUS       sequence-175070-        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  JOURNAL   Unpublished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            "Unpublished"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-Trp-eK-Flv
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     569..588
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(608..685)
                     /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(769..791)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    877..1017
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     932..951
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1032..1049)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1203..1791)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1283..1302)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1962..2753,2754..2822))
                     /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     2585..2604
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2823..2927)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2995..3013
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3037..3051,3052..3057,3058..3074,3075..3081)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3057..3073
                     /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    3089..3105
                     /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             3124..3135
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3148..3171
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3175..4272
                     /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     4246..4269
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    4342..4366
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4513..4602
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(5054..5078)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5146..5240
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5284..5943
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5350..5369)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6008..6094
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6187..6662,1..607))
                     /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 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
       61 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
      121 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
      181 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
      241 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
      301 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
      361 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
      421 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
      481 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
      541 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
      601 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
      661 aggttttgca ccattcgatg gtgtcggaat ttcgggcagc gttgggtcct ggccacgggt
      721 gcgcatgatc tagagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
      781 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
      841 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
      901 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
      961 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     1021 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1081 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1141 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1201 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1261 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1321 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1381 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1441 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1501 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     1561 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     1621 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     1681 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     1741 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     1801 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     1861 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     1921 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     1981 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2041 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2101 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2161 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2221 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2281 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2341 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     2401 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     2461 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     2521 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     2581 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     2641 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     2701 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     2761 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     2821 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     2881 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     2941 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     3001 cgtatcacga ggccctttcg tcttcacctc gagaaatcat aaaaaattta tttgctttgt
     3061 gagcggataa caattataat agattcaatt gtgagcggat aacaatttca cacagaattc
     3121 attaaagagg agaaattaac catgggacat caccatcatc accatcacca ttcaaaaatc
     3181 gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa
     3241 gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa
     3301 ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg
     3361 gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac
     3421 aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag
     3481 ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg
     3541 ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa
     3601 ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct
     3661 gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc
     3721 gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa
     3781 cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca
     3841 gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat
     3901 ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg
     3961 agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac
     4021 tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta
     4081 gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa
     4141 aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc
     4201 gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa
     4261 gacgcgcaga ctaattcgag ctcgaacaac aacaacaata acaataacaa caacctcggg
     4321 atcgacgaaa acctgtacct cacaagtttg tacaaaaaag caggcttaga aaacctgtat
     4381 tttcagtggc acggatctgg agcttggctg ttgcccgtct cactggtgaa aagaaaaacc
     4441 accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     4501 ggaatgggat cctacccata cgatgttcct gactatgcgg gctatcccta tgacgtcccg
     4561 gactatgcag gatcctatcc atacgacgtt ccagattacg ctggtgctgc agatatcact
     4621 cttatcagcg gcagcaccct cggcggtgcc gaatatgtag cagaacacct ggctgaaaag
     4681 ctggaagagg cgggttttac caccgaaacg ctgcacggtc cgctgttgga agatttatct
     4741 gcctcgggga tctggctgat tatcagctcc acccacggtg ccggagatat tccggacaac
     4801 ctttctcctt tctatgaagc attgcaggaa cagaagcccg atctttctgc ggtccgcttt
     4861 ggcgcaatcg gtattggtag tcgtgaatat gacacctttt gtggggctat cgataaactc
     4921 gaggcagaac tcaaagattc cggtgcaaaa cagacaggcg aaacactgaa gatcaacatt
     4981 cttgatcacg acattccgga agatccggca gaagaatggc tgggatcgtg gattaattta
     5041 ctcaaataat gatacccagc tttcttgtac aaagtggtga tggcgcgcct gtaggacgtc
     5101 gacggtacca tcgatacgcg ttcgaagctt aattagctga gcttggactc ctgttgatag
     5161 atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg
     5221 cgttttttat tggtgagaat ccaagctagc ttggcgagat tttcaggagc taaggaagct
     5281 aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa
     5341 gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg
     5401 gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt
     5461 attcacattc ttgcccgcct gatgaatgct catccggaat ttcgtatggc aatgaaagac
     5521 ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact
     5581 gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata
     5641 tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt
     5701 gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac
     5761 gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa
     5821 ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc
     5881 catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg
     5941 taattttttt aaggcagtta ttggtgccct taaacgcctg gggtaatgac tctctagctt
     6001 gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     6061 tttgtcggtg aacgctctcc tgagtaggac aaatccgccc tctagattac gtgcagtcga
     6121 tgataagctg tcaaacatga gaattgtgcc taatgagtga gctaacttac attaattgcg
     6181 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6241 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     6301 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     6361 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     6421 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     6481 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     6541 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     6601 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     6661 tt
//
