LOCUS       40924_46718        5977 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pWW3404, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5977)
  AUTHORS   Whitaker WR, Shepherd ES, Sonnenburg JL.
  TITLE     Tunable expression tools enable single-cell strain distinction in 
            the gut microbiome
  JOURNAL   Cell (2017) In press
REFERENCE   2  (bases 1 to 5977)
  AUTHORS   Whitaker WR, Shepherd ES, Sonnenburg JL.
  TITLE     Direct Submission
  JOURNAL   
REFERENCE   3  (bases 1 to 5977)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5977)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell (2017)
            In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (15-MAR-2017) Microbiology "
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(29..417)
                     /direction=LEFT
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     oriT            588..697
                     /label=oriT
                     /note="incP origin of transfer"
     regulatory      841..933
                     /regulatory_class="promoter"
     misc_RNA        939..989
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      1014..1036
                     /regulatory_class="ribosome_binding_site"
     CDS             1037..1750
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Pedelacq et al., 2006)"
     CDS             1757..1780
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             1781..1804
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             1805..1828
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             1832..1849
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      1872..1899
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     CDS             complement(2131..3414)
                     /codon_start=1
                     /product="NBU integrase"
                     /label=NBU integrase
                     /protein_id="ARI71333.1"
                     /translation="MNIKRNIIFALESRKKNGVPIVENVPIRMRVIFASQRIEFTTGYR
                     IDVAKWDADKQRVKSGCTNKLKQSAAEINTDLLKYYAEIQNIFKEFEVQEVMPTTQQLK
                     EAFNMRMKDTSEEQPEEAPVSFWEVFDEFVKECGNQNNWTASTYEKFAAVRNHLKEFKE
                     DATFNYFNEFGLNEYVNFLRDTKDMRNSTIGKQMGFLKWFLRWSFKKGHHQNIAYDTFK
                     PKLKTTSKKVIFLTWDELNKLKDYQIPKDKQYLERVRDVFLFCCFTSLRYSDVRNLKRS
                     DVKSDHIEITTVKTADSLTIELNKYSKAILDKYKDIHFENYMALPVISNQKMNDYLKEL
                     GELAEINEPVRETYYKGNERIDEVTPKYALLSTHAGRRTFICNALALGIPAQVVMKWTG
                     HSDYKAMKPYIDIADDIKANAMNKFNQL"
     misc_feature    3574..3585
                     /label=integration site
                     /note="integration site"
     CDS             complement(3857..4714)
                     /label=AmpR
                     /note="beta-lactamase"
     CDS             5202..5936
                     /codon_start=1
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="ARI71336.1"
                     /translation="MNKVNIKDSQNFITSKYHIEKIMNCISLDEKDNIFEIGAGKGHFT
                     AGLVKRCNFVTAIEIDSKLCEVTRNKLLNYPNYQIVNDDILKFTFPSHNPYKIFGSIPY
                     NISTNIIRKIVFESSATISYLIVEYGFAKMLLDTNRSLALLLMAEVDISILAKIPRYYF
                     HPKPKVDSTLIVLKRKPAKMAFKERKKYETFVMKWVNKEYEKLFTKNQFNKALKHARIY
                     DINNISFEQFVSLFNSYKIFNG"
ORIGIN
        1 ccgacaccgc ggtggagggg aattcccatg tcagccgtta agtgttcctg tgtcactcaa
       61 aattgctttg agaggctcta agggcttctc agtgcgttac atccctggct tgttgtccac
      121 aaccgttaaa ccttaaaagc tttaaaagcc ttatatattc ttttttttct tataaaactt
      181 aaaaccttag aggctattta agttgctgat ttatattaat tttattgttc aaacatgaga
      241 gcttagtacg tgaaacatga gagcttagta cgttagccat gagagcttag tacgttagcc
      301 atgagggttt agttcgttaa acatgagagc ttagtacgtt aaacatgaga gcttagtacg
      361 tgaaacatga gagcttagta cgtactatca acaggttgaa ctgctgatct tcagatcctc
      421 tacgccggac gcatcgtggc cggatcaatt ccgttttccg ctgcataacc ctgcttcggg
      481 gtcattatag cgattttttc ggtatatcca tcctttttcg cacgatatac aggattttgc
      541 caaagggttc gtgtagactt tccttggtgt atccaacggc gtcagccggg caggataggt
      601 gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg tcccttattc gcacctggcg
      661 gtgctcaacg ggaatcctgc tctgcgaggc tggccggcta ccgccggcgt aacagatgag
      721 ggcaagcgga tggctgatga aaccaagcca accaggaagg gcagcccacc tatcacggaa
      781 ttgatccccc tcgaattgga attcgttccg tctcgattca gatctaaagg atccaatcta
      841 gagtttgcaa tggttaatct attgttaaaa tttaaagttt cacttgaact ttcaaataat
      901 gttcttatat ttgcagtgtc gaaagaaaca aaggactagc tgtcaccgga tgtgctttcc
      961 ggtctgatga gtccgtgagg acgaaacagc ctctacaaat aattttgttt aacacacaac
     1021 atttaaaaaa taacaaatgc gtaaaggcga agagctgttc actggtgtcg tccctattct
     1081 ggtggaactg gatggtgatg tcaacggtca taagttttcc gtgcgtggcg agggtgaagg
     1141 tgacgcaact aatggtaaac tgacgctgaa gttcatctgt actactggta aactgccggt
     1201 tccttggccg actctggtaa cgacgctgac ttatggtgtt cagtgctttg ctcgttatcc
     1261 ggaccatatg aagcagcatg acttcttcaa gtccgccatg ccggaaggct atgtgcagga
     1321 acgcacgatt tcctttaagg atgacggcac gtacaaaacg cgtgcggaag tgaaatttga
     1381 aggcgatacc ctggtaaacc gcattgagct gaaaggcatt gactttaaag aagacggcaa
     1441 tatcctgggc cataagctgg aatacaattt taacagccac aatgtttaca tcaccgccga
     1501 taaacaaaaa aatggcatta aagcgaattt taaaattcgc cacaacgtgg aggatggcag
     1561 cgtgcagctg gctgatcact accagcaaaa cactccaatc ggtgatggtc ctgttctgct
     1621 gccagacaat cactatctga gcacgcaaag cgttctgtct aaagatccga acgagaaacg
     1681 cgatcatatg gttctgctgg agttcgtaac cgcagcgggc atcacgcatg gtatggatga
     1741 actgtacaaa ggatctgatt acaaggacga tgatgataag gactacaaag atgatgatga
     1801 caaagattat aaggacgatg atgataaagg tcatcatcat catcatcact aaatcctagc
     1861 tgattagctc gagaaggcca tcctgacgga tggccttttt tttgtatcga gacgactagt
     1921 gtaattgcct atcttccagt gatggaacag catttgtgca ttggctgcaa caatcagcct
     1981 tacttgtgcc tgttctattt ccgaaccgac cgcttgtatg aatccatcaa aattcgtttt
     2041 ctctatgttg gattccttgt tgctcatatt gtgatgataa tttctacaaa tatagtcatt
     2101 ggtaactatc tatgaaactg tttgatactt ttatagttga ttaaacttgt tcatggcatt
     2161 tgccttaata tcatccgcta tgtcaatgta gggtttcata gctttgtagt cgctgtgtcc
     2221 cgtccatttc atgaccacct gtgccgggat tccgagagcc agcgcattgc agatgaatgt
     2281 ccttcttcct gcatgggtac tgagcaaagc gtatttgggt gtgacttcat caatacgttc
     2341 atttcccttg tagtaggttt cccgtacagg ctcgttgatt tctgccagtt cgcccagctc
     2401 tttcaggtaa tcgttcatct tctggttgct gatgacgggc agagccatgt aattctcgaa
     2461 atggatgtcc ttgtatttgt ccagtatggc tttgctgtat ttgttcagtt caatcgtcag
     2521 gctgtcggca gtcttgactg tggttatttc gatgtggtcg gacttcacat cgcttctttt
     2581 cagattgcga acatccgaat accgcaaact cgtaaagcag cagaacagga aaacatcacg
     2641 cacacgttcc aggtattgct tatccttggg tatctggtag tctttcagct tgttcagttc
     2701 atcccaagtc aggaagatta cttttttcga ggtggttttc agtttcggtt tgaacgtatc
     2761 gtatgcaatg ttctgatgat gtcctttctt gaagctccag cgcaggaacc atttgaggaa
     2821 tcccatttgc ttgccgatgg tgctgtttct catatccttg gtgtcacgca ggaagttgac
     2881 gtattcgttc aatccaaact cgttgaaata gttgaacgtt gcatcctcct tgaactcttt
     2941 gaggtggttc ctcactgctg caaatttttc ataggtggat gccgtccagt tattctggtt
     3001 accgcactct tttacaaact catcgaacac ctcccaaaag ctgacagggg cttcttccgg
     3061 ctgttcttcg ctggtgtctt tcattctcat gttgaaagct tccttcaact gttgggtcgt
     3121 tggcatgacc tcctgcacct caaattcctt gaaaatattc tggatttcgg catagtattt
     3181 cagcaagtcc gtattgattt cggctgcact ttgctttagc ttgttggtac atccgctctt
     3241 tacccgctgc ttatctgcat cccatttggc tacgtcaatc cggtagcccg ttgtaaactc
     3301 gatgcgttgg ctggcaaaga tgacacgcat acggatgggt acgttctcta cgattggcac
     3361 accgttcttt ttccggctct ccaatgcaaa aatgatgttg cgcttgatat tcataattgg
     3421 gtgcgtttga aattctacac ccaaatatac acccaattat tgagatagca aaagacattt
     3481 agaaacattt acttttactc tatattgtaa tttacacttg attatcagtc gtttgcagtc
     3541 ttatgatatt ctgtgaaagt ataagttcga gagcctgtct ctccgcaaaa aacgctgaaa
     3601 atcagcagat tgcaaaacaa acaccctgtt ttacacccaa gaatgtaaag tcggctgttt
     3661 ttgttttatt taagataata caaccactac ataataaaag agtagcgata ttaaaagaat
     3721 ccgatgagaa aagactaata tttatctatc cattcagttt gatttttcag gactttacat
     3781 cgtcctgaaa gtatttgttg gtaccgagga cgcgacagct gcagacgcgt tatgagtaaa
     3841 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3901 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3961 taccatctgg ccccagtgct gcaatgatac cgcgggaccc acgctcaccg gctccagatt
     4021 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4081 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4141 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4201 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4261 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4321 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4381 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4441 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     4501 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     4561 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     4621 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4681 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tcatggccgc
     4741 gggattaaaa gtcggggatt ggtgaacaaa aaggtgtttc tctctttaag agaaatatcg
     4801 ttttgctaaa cagttgatat tgaggtatca ttttatcgta aaagacattt ttgctcaaca
     4861 attgcttgac ggaaatcaac aaattttagc attttgtaaa aaagtcgcta tataatttgg
     4921 tgaattggag ttattttcat atttttgcat cccgaagagt ttctcttaaa gagagaaaca
     4981 tcttttgcat accttttccg accgaatttt tatgtcgtaa agaggggctt tgcagggggt
     5041 ggactcagaa agatgagaat agatgactat tgtagttgaa acacatagaa agttgctgat
     5101 atacagaccg atacgcatat cgggatgaac catgagtacg ttcttttctc aaaaaacata
     5161 aatattcgaa aagagatgca ataaattaag gagaggttat aatgaacaaa gtaaatataa
     5221 aagatagtca aaattttatt acttcaaaat atcacataga aaaaataatg aattgcataa
     5281 gtttagatga aaaagataac atctttgaaa taggtgcagg gaaaggtcat tttactgctg
     5341 gattggtaaa gagatgtaat tttgtaacgg cgatagaaat tgattctaaa ttatgtgagg
     5401 taactcgtaa taagctctta aattatccta actatcaaat agtaaatgat gatatactga
     5461 aatttacatt tcctagccac aatccatata aaatatttgg cagcatacct tacaacataa
     5521 gcacaaatat aattcgaaaa attgtttttg aaagttcagc cacaataagt tatttaatag
     5581 tggaatatgg ttttgctaaa atgttattag atacaaacag atcactagca ttgctgttaa
     5641 tggcagaggt agatatttct atattagcaa aaattcctag gtattatttc catccaaaac
     5701 ctaaagtgga tagcacatta attgtattaa aaagaaagcc agcaaaaatg gcatttaaag
     5761 agagaaaaaa atatgaaact tttgtaatga aatgggttaa caaagagtac gaaaaactgt
     5821 ttacaaaaaa tcaatttaat aaagctttaa aacatgcgag aatatatgat ataaacaata
     5881 ttagtttcga acaatttgta tcgctattta atagttataa aatatttaac ggctaaaaac
     5941 aataggccac atgcaactgt aaatgtttac gcgggta
//
