LOCUS       sequence-173972-        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  JOURNAL   Unpublished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            "Unpublished"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-Phe-eK-Flv
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     97..120
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    193..217
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             364..453
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(905..929)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      997..1091
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1135..1794
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1201..1220)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1859..1945
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2038..3120)
                     /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"
     primer_bind     3082..3101
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3121..3198)
                     /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(3282..3304)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3390..3530
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3445..3464
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3545..3562)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3716..4304)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3796..3815)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4475..5266,5267..5335))
                     /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     5098..5117
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5336..5440)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5508..5526
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5550..5564,5565..5570,5571..5587,5588..5594)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    5570..5586
                     /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    5602..5618
                     /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             5637..5648
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             5661..5684
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             join(5688..6662,1..123)
                     /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"
ORIGIN
        1 aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact
       61 gcggtgatca acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag
      121 actaattcga gctcgaacaa caacaacaat aacaataaca acaacctcgg gatcgacgaa
      181 aacctgtacc tcacaagttt gtacaaaaaa gcaggcttag agaatcttta ttttcagttc
      241 cacggatctg gagcttggct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
      301 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gggaatggga
      361 tcctacccat acgatgttcc tgactatgcg ggctatccct atgacgtccc ggactatgca
      421 ggatcctatc catacgacgt tccagattac gctggtgctg cagatatcac tcttatcagc
      481 ggcagcaccc tcggcggtgc cgaatatgta gcagaacacc tggctgaaaa gctggaagag
      541 gcgggtttta ccaccgaaac gctgcacggt ccgctgttgg aagatttatc tgcctcgggg
      601 atctggctga ttatcagctc cacccacggt gccggagata ttccggacaa cctttctcct
      661 ttctatgaag cattgcagga acagaagccc gatctttctg cggtccgctt tggcgcaatc
      721 ggtattggta gtcgtgaata tgacaccttt tgtggggcta tcgataaact cgaggcagaa
      781 ctcaaagatt ccggtgcaaa acagacaggc gaaacactga agatcaacat tcttgatcac
      841 gacattccgg aagatccggc agaagaatgg ctgggatcgt ggattaattt actcaaataa
      901 tgatacccag ctttcttgta caaagtggtg atggcgcgcc tgtaggacgt cgacggtacc
      961 atcgatacgc gttcgaagct taattagctg agcttggact cctgttgata gatccagtaa
     1021 tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg gcgtttttta
     1081 ttggtgagaa tccaagctag cttggcgaga ttttcaggag ctaaggaagc taaaatggag
     1141 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
     1201 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
     1261 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
     1321 cttgcccgcc tgatgaatgc tcatccggaa tttcgtatgg caatgaaaga cggtgagctg
     1381 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
     1441 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     1501 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     1561 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     1621 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     1681 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
     1741 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt
     1801 taaggcagtt attggtgccc ttaaacgcct ggggtaatga ctctctagct tgaggcatca
     1861 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     1921 gaacgctctc ctgagtagga caaatccgcc ctctagatta cgtgcagtcg atgataagct
     1981 gtcaaacatg agaattgtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     2041 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     2101 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     2161 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     2221 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     2281 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     2341 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     2401 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     2461 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     2521 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     2581 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     2641 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     2701 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     2761 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     2821 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     2881 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     2941 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     3001 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     3061 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     3121 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     3181 gcaccattcg atggtgtcgg aatttcgggc agcgttgggt cctggccacg ggtgcgcatg
     3241 atctagagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     3301 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     3361 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     3421 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     3481 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     3541 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3601 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3661 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3721 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3781 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3841 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3901 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3961 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4021 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4081 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4141 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4201 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4261 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4321 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4381 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4441 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4501 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4561 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4621 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4681 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4741 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4801 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4861 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4921 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4981 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5041 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5101 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5161 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5221 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5281 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5341 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5401 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5461 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     5521 cgaggccctt tcgtcttcac ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga
     5581 taacaattat aatagattca attgtgagcg gataacaatt tcacacagaa ttcattaaag
     5641 aggagaaatt aaccatggga catcaccatc atcaccatca ccattcaaaa atcgaagaag
     5701 gtaaactggt aatctggatt aacggcgata aaggctataa cggtctcgct gaagtcggta
     5761 agaaattcga gaaagatacc ggaattaaag tcaccgttga gcatccggat aaactggaag
     5821 agaaattccc acaggttgcg gcaactggcg atggccctga cattatcttc tgggcacacg
     5881 accgctttgg tggctacgct caatctggcc tgttggctga aatcaccccg gacaaagcgt
     5941 tccaggacaa gctgtatccg tttacctggg atgccgtacg ttacaacggc aagctgattg
     6001 cttacccgat cgctgttgaa gcgttatcgc tgatttataa caaagatctg ctgccgaacc
     6061 cgccaaaaac ctgggaagag atcccggcgc tggataaaga actgaaagcg aaaggtaaga
     6121 gcgcgctgat gttcaacctg caagaaccgt acttcacctg gccgctgatt gctgctgacg
     6181 ggggttatgc gttcaagtat gaaaacggca agtacgacat taaagacgtg ggcgtggata
     6241 acgctggcgc gaaagcgggt ctgaccttcc tggttgacct gattaaaaac aaacacatga
     6301 atgcagacac cgattactcc atcgcagaag ctgcctttaa taaaggcgaa acagcgatga
     6361 ccatcaacgg cccgtgggca tggtccaaca tcgacaccag caaagtgaat tatggtgtaa
     6421 cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag
     6481 gtattaacgc cgccagtccg aacaaagagc tggcaaaaga gttcctcgaa aactatctgc
     6541 tgactgatga aggtctggaa gcggttaata aagacaaacc gctgggtgcc gtagcgctga
     6601 agtcttacga ggaagagttg gcgaaagatc cacgtattgc cgccactatg gaaaacgccc
     6661 ag
//
