LOCUS       sequence-175065-        6662 bp    DNA     circular SYN 15-APR-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  JOURNAL   Unpublished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 Apr 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            "Unpublished"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-His-eK-Flv
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             74..163
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(615..639)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      707..801
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             845..1504
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(911..930)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1569..1655
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1748..2830)
                     /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     2792..2811
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2831..2908)
                     /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(2992..3014)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3100..3240
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3155..3174
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3255..3272)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3426..4014)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3506..3525)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4185..4976,4977..5045))
                     /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     4808..4827
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5046..5150)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5218..5236
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5260..5274,5275..5280,5281..5297,5298..5304)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    5280..5296
                     /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    5312..5328
                     /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             5347..5358
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             5371..5394
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             5398..6495
                     /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     6469..6492
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    6565..6589
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
       61 gggaatggga tcctacccat acgatgttcc tgactatgcg ggctatccct atgacgtccc
      121 ggactatgca ggatcctatc catacgacgt tccagattac gctggtgctg cagatatcac
      181 tcttatcagc ggcagcaccc tcggcggtgc cgaatatgta gcagaacacc tggctgaaaa
      241 gctggaagag gcgggtttta ccaccgaaac gctgcacggt ccgctgttgg aagatttatc
      301 tgcctcgggg atctggctga ttatcagctc cacccacggt gccggagata ttccggacaa
      361 cctttctcct ttctatgaag cattgcagga acagaagccc gatctttctg cggtccgctt
      421 tggcgcaatc ggtattggta gtcgtgaata tgacaccttt tgtggggcta tcgataaact
      481 cgaggcagaa ctcaaagatt ccggtgcaaa acagacaggc gaaacactga agatcaacat
      541 tcttgatcac gacattccgg aagatccggc agaagaatgg ctgggatcgt ggattaattt
      601 actcaaataa tgatacccag ctttcttgta caaagtggtg atggcgcgcc tgtaggacgt
      661 cgacggtacc atcgatacgc gttcgaagct taattagctg agcttggact cctgttgata
      721 gatccagtaa tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg
      781 gcgtttttta ttggtgagaa tccaagctag cttggcgaga ttttcaggag ctaaggaagc
      841 taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa
      901 agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct
      961 ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt
     1021 tattcacatt cttgcccgcc tgatgaatgc tcatccggaa tttcgtatgg caatgaaaga
     1081 cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac
     1141 tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat
     1201 atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat
     1261 tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa
     1321 cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca
     1381 aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt
     1441 ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc
     1501 gtaatttttt taaggcagtt attggtgccc ttaaacgcct ggggtaatga ctctctagct
     1561 tgaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     1621 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc ctctagatta cgtgcagtcg
     1681 atgataagct gtcaaacatg agaattgtgc ctaatgagtg agctaactta cattaattgc
     1741 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1801 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     1861 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     1921 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     1981 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     2041 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     2101 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     2161 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     2221 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     2281 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     2341 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     2401 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     2461 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     2521 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     2581 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     2641 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     2701 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     2761 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     2821 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     2881 gaaaggtttt gcaccattcg atggtgtcgg aatttcgggc agcgttgggt cctggccacg
     2941 ggtgcgcatg atctagagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     3001 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     3061 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     3121 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     3181 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     3241 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3301 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3361 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3421 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3481 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3541 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3601 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3661 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3721 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3781 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3841 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     3901 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3961 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4021 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4081 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4141 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4201 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4261 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4321 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4381 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4441 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4501 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4561 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4621 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4681 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4741 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4801 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4861 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4921 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4981 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5041 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5101 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5161 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     5221 aggcgtatca cgaggccctt tcgtcttcac ctcgagaaat cataaaaaat ttatttgctt
     5281 tgtgagcgga taacaattat aatagattca attgtgagcg gataacaatt tcacacagaa
     5341 ttcattaaag aggagaaatt aaccatggga catcaccatc atcaccatca ccattcaaaa
     5401 atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa cggtctcgct
     5461 gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga gcatccggat
     5521 aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga cattatcttc
     5581 tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga aatcaccccg
     5641 gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg ttacaacggc
     5701 aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa caaagatctg
     5761 ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga actgaaagcg
     5821 aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg gccgctgatt
     5881 gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat taaagacgtg
     5941 ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct gattaaaaac
     6001 aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa taaaggcgaa
     6061 acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag caaagtgaat
     6121 tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt cgttggcgtg
     6181 ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcaaaaga gttcctcgaa
     6241 aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc gctgggtgcc
     6301 gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc cgccactatg
     6361 gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc tttctggtat
     6421 gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga tgaagccctg
     6481 aaagacgcgc agactaattc gagctcgaac aacaacaaca ataacaataa caacaacctc
     6541 gggatcgacg aaaacctgta cctcacaagt ttgtacaaaa aagcaggctt agaaaacctg
     6601 tattttcagc atcacggatc tggagcttgg ctgttgcccg tctcactggt gaaaagaaaa
     6661 ac
//
