LOCUS       sequence-175072-        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  JOURNAL   Unpublished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            "Unpublished"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-Leu-e-Delta-K-Flv
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             44..133
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(585..609)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      677..771
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             815..1474
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(881..900)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1539..1625
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1718..2800)
                     /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     2762..2781
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2801..2878)
                     /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(2962..2984)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3070..3210
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3125..3144
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3225..3242)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3396..3984)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3476..3495)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4155..4946,4947..5015))
                     /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     4778..4797
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5016..5120)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5188..5206
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5230..5244,5245..5250,5251..5267,5268..5274)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    5250..5266
                     /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    5282..5298
                     /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             5317..5328
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             5341..5364
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             5368..6465
                     /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     6439..6462
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    6535..6559
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 tccccgcgcg ttggccgatt cattaatgca gggaatggga tcctacccat acgatgttcc
       61 tgactatgcg ggctatccct atgacgtccc ggactatgca ggatcctatc catacgacgt
      121 tccagattac gctggtgctg cagatatcac tcttatcagc ggcagcaccc tcggcggtgc
      181 cgaatatgta gcagaacacc tggctgaaaa gctggaagag gcgggtttta ccaccgaaac
      241 gctgcacggt ccgctgttgg aagatttatc tgcctcgggg atctggctga ttatcagctc
      301 cacccacggt gccggagata ttccggacaa cctttctcct ttctatgaag cattgcagga
      361 acagaagccc gatctttctg cggtccgctt tggcgcaatc ggtattggta gtcgtgaata
      421 tgacaccttt tgtggggcta tcgataaact cgaggcagaa ctcaaagatt ccggtgcaaa
      481 acagacaggc gaaacactga agatcaacat tcttgatcac gacattccgg aagatccggc
      541 agaagaatgg ctgggatcgt ggattaattt actcaaataa tgatacccag ctttcttgta
      601 caaagtggtg atggcgcgcc tgtaggacgt cgacggtacc atcgatacgc gttcgaagct
      661 taattagctg agcttggact cctgttgata gatccagtaa tgacctcaga actccatctg
      721 gatttgttca gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tccaagctag
      781 cttggcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
      841 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc
      901 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa
      961 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
     1021 tcatccggaa tttcgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca
     1081 cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata
     1141 ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga
     1201 aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc
     1261 ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc
     1321 cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat
     1381 tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca
     1441 acagtactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc
     1501 ttaaacgcct ggggtaatga ctctctagct tgaggcatca aataaaacga aaggctcagt
     1561 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
     1621 caaatccgcc ctctagatta cgtgcagtcg atgataagct gtcaaacatg agaattgtgc
     1681 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1741 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1801 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     1861 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     1921 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     1981 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     2041 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     2101 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     2161 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     2221 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     2281 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     2341 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     2401 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     2461 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     2521 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     2581 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     2641 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     2701 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     2761 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     2821 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcaccattcg atggtgtcgg
     2881 aatttcgggc agcgttgggt cctggccacg ggtgcgcatg atctagagct gcctcgcgcg
     2941 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     3001 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     3061 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac
     3121 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
     3181 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
     3241 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3301 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3361 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3421 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3481 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3541 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3601 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3661 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3721 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     3781 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     3841 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     3901 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     3961 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4021 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4081 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4141 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4201 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4261 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4321 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4381 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4441 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     4501 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4561 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4621 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4681 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4741 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     4801 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4861 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     4921 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     4981 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5041 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5101 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     5161 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcac
     5221 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
     5281 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aaccatggga
     5341 catcaccatc atcaccatca ccattcaaaa atcgaagaag gtaaactggt aatctggatt
     5401 aacggcgata aaggctataa cggtctcgct gaagtcggta agaaattcga gaaagatacc
     5461 ggaattaaag tcaccgttga gcatccggat aaactggaag agaaattccc acaggttgcg
     5521 gcaactggcg atggccctga cattatcttc tgggcacacg accgctttgg tggctacgct
     5581 caatctggcc tgttggctga aatcaccccg gacaaagcgt tccaggacaa gctgtatccg
     5641 tttacctggg atgccgtacg ttacaacggc aagctgattg cttacccgat cgctgttgaa
     5701 gcgttatcgc tgatttataa caaagatctg ctgccgaacc cgccaaaaac ctgggaagag
     5761 atcccggcgc tggataaaga actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg
     5821 caagaaccgt acttcacctg gccgctgatt gctgctgacg ggggttatgc gttcaagtat
     5881 gaaaacggca agtacgacat taaagacgtg ggcgtggata acgctggcgc gaaagcgggt
     5941 ctgaccttcc tggttgacct gattaaaaac aaacacatga atgcagacac cgattactcc
     6001 atcgcagaag ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg cccgtgggca
     6061 tggtccaaca tcgacaccag caaagtgaat tatggtgtaa cggtactgcc gaccttcaag
     6121 ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg
     6181 aacaaagagc tggcaaaaga gttcctcgaa aactatctgc tgactgatga aggtctggaa
     6241 gcggttaata aagacaaacc gctgggtgcc gtagcgctga agtcttacga ggaagagttg
     6301 gcgaaagatc cacgtattgc cgccactatg gaaaacgccc agaaaggtga aatcatgccg
     6361 aacatcccgc agatgtccgc tttctggtat gccgtgcgta ctgcggtgat caacgccgcc
     6421 agcggtcgtc agactgtcga tgaagccctg aaagacgcgc agactaattc gagctcgaac
     6481 aacaacaaca ataacaataa caacaacctc gggatcgacg aaaacctgta cctcacaagt
     6541 ttgtacaaaa aagcaggctt agagaatctt tattttcagt tgcacggatc tggagcttgg
     6601 ctgttgcccg tctcactggt gagaagaaga accaccctgg cgcccaatac gcaaaccgcc
     6661 tc
//
