LOCUS       sequence-175060-        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  JOURNAL   Unpublished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            "Unpublished"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-Leu-eK-Flv
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     859..878
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(898..975)
                     /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(1059..1081)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1167..1307
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1222..1241
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1322..1339)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1493..2081)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1573..1592)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2252..3043,3044..3112))
                     /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     2875..2894
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3113..3217)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3285..3303
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3327..3341,3342..3347,3348..3364,3365..3371)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3347..3363
                     /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    3379..3395
                     /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             3414..3425
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3438..3461
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3465..4562
                     /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     4536..4559
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    4632..4656
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4803..4892
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(5344..5368)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5436..5530
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5574..6233
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5640..5659)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6298..6384
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6477..6662,1..897))
                     /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 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
       61 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
      121 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
      181 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
      241 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
      301 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
      361 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
      421 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
      481 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
      541 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
      601 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
      661 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
      721 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
      781 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
      841 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
      901 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca
      961 ccattcgatg gtgtcggaat ttcgggcagc gttgggtcct ggccacgggt gcgcatgatc
     1021 tagagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     1081 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     1141 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     1201 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     1261 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     1321 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     1381 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     1441 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     1501 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     1561 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1621 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     1681 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     1741 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1801 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     1861 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     1921 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     1981 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2041 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2101 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2161 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     2221 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     2281 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     2341 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     2401 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     2461 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     2521 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     2581 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     2641 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     2701 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     2761 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     2821 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     2881 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     2941 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3001 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3061 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3121 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     3181 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     3241 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     3301 ggccctttcg tcttcacctc gagaaatcat aaaaaattta tttgctttgt gagcggataa
     3361 caattataat agattcaatt gtgagcggat aacaatttca cacagaattc attaaagagg
     3421 agaaattaac catgggacat caccatcatc accatcacca ttcaaaaatc gaagaaggta
     3481 aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga
     3541 aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga
     3601 aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc
     3661 gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc
     3721 aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt
     3781 acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc
     3841 caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg
     3901 cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg
     3961 gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg
     4021 ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg
     4081 cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca
     4141 tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg
     4201 tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta
     4261 ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga
     4321 ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt
     4381 cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa aacgcccaga
     4441 aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg
     4501 cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga
     4561 ctaattcgag ctcgaacaac aacaacaata acaataacaa caacctcggg atcgacgaaa
     4621 acctgtacct cacaagtttg tacaaaaaag caggcttaga gaatctttat tttcagttgc
     4681 acggatctgg agcttggctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     4741 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ggaatgggat
     4801 cctacccata cgatgttcct gactatgcgg gctatcccta tgacgtcccg gactatgcag
     4861 gatcctatcc atacgacgtt ccagattacg ctggtgctgc agatatcact cttatcagcg
     4921 gcagcaccct cggcggtgcc gaatatgtag cagaacacct ggctgaaaag ctggaagagg
     4981 cgggttttac caccgaaacg ctgcacggtc cgctgttgga agatttatct gcctcgggga
     5041 tctggctgat tatcagctcc acccacggtg ccggagatat tccggacaac ctttctcctt
     5101 tctatgaagc attgcaggaa cagaagcccg atctttctgc ggtccgcttt ggcgcaatcg
     5161 gtattggtag tcgtgaatat gacacctttt gtggggctat cgataaactc gaggcagaac
     5221 tcaaagattc cggtgcaaaa cagacaggcg aaacactgaa gatcaacatt cttgatcacg
     5281 acattccgga agatccggca gaagaatggc tgggatcgtg gattaattta ctcaaataat
     5341 gatacccagc tttcttgtac aaagtggtga tggcgcgcct gtaggacgtc gacggtacca
     5401 tcgatacgcg ttcgaagctt aattagctga gcttggactc ctgttgatag atccagtaat
     5461 gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg cgttttttat
     5521 tggtgagaat ccaagctagc ttggcgagat tttcaggagc taaggaagct aaaatggaga
     5581 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
     5641 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
     5701 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
     5761 ttgcccgcct gatgaatgct catccggaat ttcgtatggc aatgaaagac ggtgagctgg
     5821 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
     5881 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     5941 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     6001 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     6061 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     6121 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     6181 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg taattttttt
     6241 aaggcagtta ttggtgccct taaacgcctg gggtaatgac tctctagctt gaggcatcaa
     6301 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     6361 aacgctctcc tgagtaggac aaatccgccc tctagattac gtgcagtcga tgataagctg
     6421 tcaaacatga gaattgtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     6481 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     6541 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     6601 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     6661 ct
//
