LOCUS       40924_27414        5681 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pL0E_4-R, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5681)
  AUTHORS   Hochrein L, Machens F, Gremmels J, Schulz K, Messerschmidt K, 
            Mueller-Roeber B.
  TITLE     AssemblX: a user-friendly toolkit for rapid and reliable multi-gene 
            assemblies
  JOURNAL   Nucleic Acids Res. (2017) In press
  PUBMED    28130422
REFERENCE   2  (bases 1 to 5681)
  AUTHORS   Hochrein L, Machens F, Gremmels J, Schulz K, Messerschmidt K, 
            Mueller-Roeber B.
  TITLE     Direct Submission
  JOURNAL   Submitted (11-NOV-2016) Department of Molecular Biology - Cell2Fab 
            research unit, University of Potsdam, Karl-Liebknecht-Str. 24-25, 
            Potsdam, Brandenburg 14476, Germany
REFERENCE   3  (bases 1 to 5681)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5681)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. (2017) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (11-NOV-2016) Department of Molecular Biology - Cell2Fab research 
            unit, University of Potsdam, Karl-Liebknecht-Str. 24-25, Potsdam, 
            Brandenburg 14476, Germany"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(582..1382)
                     /codon_start=1
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(1383..1603)
                     /label=URA3 promoter
     rep_origin      complement(2202..3082)
                     /direction=LEFT
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(3431..4222)
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHDDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(4223..4327)
                     /label=AmpR promoter
     misc_feature    4396..4435
                     /function="8 cutter set: AscI, SbfI, SwaI, FseI, PmeI"
                     /label=8 cutter set: SwaI, AscI, PmeI, SbfI, FseI
                     /note="8 cutter set: SwaI, AscI, PmeI, SbfI, FseI"
     misc_feature    4436..4471
                     /label=homology region E4*
                     /note="homology region E4*"
     CDS             4807..5109
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     promoter        5147..5427
                     /label=TRP1 promoter
     misc_feature    5428..5478
                     /label=homology region ER*
                     /note="homology region ER*"
     misc_feature    5482..5521
                     /label=8 cutter set: SwaI, AscI, PmeI, SbfI, FseI
                     /note="8 cutter set: SwaI, AscI, PmeI, SbfI, FseI"
     rep_origin      5547..5681
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
       61 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
      121 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
      181 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
      241 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
      301 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
      361 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
      421 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
      481 gttcctggcc ttttgctggc cttttgctca tgatataatt aaattgaagc tctaatttgt
      541 gagtttagta tacatgcatt tacttataat acagtttttt agttttgctg gccgcatctt
      601 ctcaaatatg cttcccagcc tgcttttctg taacgttcac cctctacctt agcatccctt
      661 ccctttgcaa atagtcctct tccaacaata ataatgtcag atcctgtaga gaccacatca
      721 tccacggttc tatactgttg acccaatgcg tctcccttgt catctaaacc cacaccgggt
      781 gtcataatca accaatcgta accttcatct cttccaccca tgtctctttg agcaataaag
      841 ccgataacaa aatctttgtc gctcttcgca atgtcaacag tacccttagt atattctcca
      901 gtagataggg agcccttgca tgacaattct gctaacatca aaaggcctct aggttccttt
      961 gttacttctt ctgccgcctg cttcaaaccg ctaacaatac ctgggcccac cacaccgtgt
     1021 gcattcgtaa tgtctgccca ttctgctatt ctgtatacac ccgcagagta ctgcaatttg
     1081 actgtattac caatgtcagc aaattttctg tcttcgaaga gtaaaaaatt gtacttggcg
     1141 gataatgcct ttagcggctt aactgtgccc tccatggaaa aatcagtcaa gatatccaca
     1201 tgtgttttta gtaaacaaat tttgggacct aatgcttcaa ctaactccag taattccttg
     1261 gtggtacgaa catccaatga agcacacaag tttgtttgct tttcgtgcat gatattaaat
     1321 agcttggcag caacaggact aggatgagta gcagcacgtt ccttatatgt agctttcgac
     1381 atgatttatc ttcgtttcct gcaggttttt gttctgtgca gttgggttaa gaatactggg
     1441 caatttcatg tttcttcaac actacatatg cgtatatata ccaatctaag tctgtgctcc
     1501 ttccttcgtt cttccttctg ttcggagatt accgaatcaa aaaaatttca aagaaaccga
     1561 aatcaaaaaa aagaataaaa aaaaaatgat gaattgaatt gaaaagctag cttatcgatg
     1621 ataagctgtc aaagatgaga attaattcca cggactatag actatactag atactccgtc
     1681 tactgtacga tacacttccg ctcaggtcct tgtcctttaa cgaggcctta ccactctttt
     1741 gttactctat tgatccagct cagcaaaggc agtgtgatct aagattctat cttcgcgatg
     1801 tagtaaaact agctagaccg agaaagagac tagaaatgca aaaggcactt ctacaatggc
     1861 tgccatcatt attatccgat gtgacgctgc agcttctcaa tgatattcga atacgctttg
     1921 aggagataca gcctaatatc cgacaaactg ttttacagat ttacgatcgt acttgttacc
     1981 catcattgaa ttttgaacat ccgaacctgg gagttttccc tgaaacagat agtatatttg
     2041 aacctgtata ataatatata gtctagcgct ttacggaaga caatgtatgt atttcggttc
     2101 ctggagaaac tattgcatct attgcatagg taatcttgca cgtcgcatcc ccggttcatt
     2161 ttctgcgttt ccatcttgca cttcaatagc atatctttgt taacgaagca tctgtgcttc
     2221 attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct
     2281 gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg
     2341 cttcattttt gtaaaacaaa aatgcaacgc gacgagagcg ctaatttttc aaacaaagaa
     2401 tctgagctgc atttttacag aacagaaatg caacgcgaga gcgctatttt accaacaaag
     2461 aatctatact tcttttttgt tctacaaaaa tgcatcccga gagcgctatt tttctaacaa
     2521 agcatcttag attacttttt ttctcctttg tgcgctctat aatgcagtct cttgataact
     2581 ttttgcactg taggtccgtt aaggttagaa gaaggctact ttggtgtcta ttttctcttc
     2641 cataaaaaaa gcctgactcc acttcccgcg tttactgatt actagcgaag ctgcgggtgc
     2701 attttttcaa gataaaggca tccccgatta tattctatac cgatgtggat tgcgcatact
     2761 ttgtgaacag aaagtgatag cgttgatgat tcttcattgg tcagaaaatt atgaacggtt
     2821 tcttctattt tgtctctata tactacgtat aggaaatgtt tacattttcg tattgttttc
     2881 gattcactct atgaatagtt cttactacaa tttttttgtc taaagagtaa tactagagat
     2941 aaacataaaa aatgtagagg tcgagtttag atgcaagttc aaggagcgaa aggtggatgg
     3001 gtaggttata tagggatata gcacagagat atatagcaaa gagatacttt tgagcaatgt
     3061 ttgtggaagc ggtattcgca atgggaagct ccaccccggt tgataatcag aaaagcccca
     3121 aaaacaggaa gattgtataa gcaaatattt aaattgtaat tctttcctgc gttatcccct
     3181 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3241 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agggtctgac gctcagtgga
     3301 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3361 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3421 ctgacagtca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag
     3481 cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa
     3541 tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt
     3601 cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgtcat
     3661 gggtcacgac gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg
     3721 ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca
     3781 tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg
     3841 gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag
     3901 caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc
     3961 ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg
     4021 atagccgcgc tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa
     4081 aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg
     4141 tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt
     4201 gcaatccatc ttgttcaatc atactcttcc tttttcaata ttattgaagc atttatcagg
     4261 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     4321 ttccgcgcac atttccccga aaagtgccac ctgacgtcgg acggatcgct tgcctgtaac
     4381 ttacacgcgc ctcgtggcgc gcccctgcag gatttaaatg gccggccgtt taaactagtg
     4441 atagcgttca atacttcctg gcacgataat aaagcttggc ttactaaaag ccagataaca
     4501 gtatgcgtat ttgcgcgctg atttttgcgg tataagaata tatactgata tgtatacccg
     4561 aagtatgtca aaaagaggta tgctatgaag cagcgtatta cagtgacagt tgacagcgac
     4621 agctatcagt tgctcaaggc atatatgatg tcaatatctc cggtctggta agcacaacca
     4681 tgcagaatga agcccgtcgt ctgcgtgccg aacgctggaa agcggaaaat caggaaggga
     4741 tggctgaggt cgcccggttt attgaaatga acggctcttt tgctgacgag aacaggggct
     4801 ggtgaaatgc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg
     4861 gatgtacaga gtgatattat tgacacgccc gggcgacgga tggtgatccc cctggccagt
     4921 gcacgtctgc tgtcagataa agtcccccgt gaactttacc cggtggtgca tatcggggat
     4981 gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa
     5041 gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc
     5101 tggggaatat aaatgtcagg ctcccttata cacagccagt aagcttaacg acattactat
     5161 atatataata taggaagcat ttaatagaca gcatcgtaat atatgtgtac tttgcagtta
     5221 tgacgccaga tggcagtagt ggaagatatt ctttattgaa aaatagcttg tcaccttacg
     5281 tacaatcttg atccggagct tttctttttt tgccgattaa gaattaattc ggtcgaaaaa
     5341 agaaaaggag agggccaaga gggagggcat tggtgactat tgagcacgtg agtatacgtg
     5401 attaagcaca caaaggcagc ttggagtatg tctgttatta atttcacagg tagttctggt
     5461 ccattggtga aagtttgctg aatttaaatg gcgcgccgtt taaaccctgc aggggccggc
     5521 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5581 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5641 actctttttc cgaaggtaac tggcttcagc agagcgcaga t
//
