LOCUS       40924_46733        6031 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pWW3452, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6031)
  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 6031)
  AUTHORS   Whitaker WR, Shepherd ES, Sonnenburg JL.
  TITLE     Direct Submission
  JOURNAL   
REFERENCE   3  (bases 1 to 6031)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6031)
  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..6031
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(25..413)
                     /direction=LEFT
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     oriT            584..693
                     /label=oriT
                     /note="incP origin of transfer"
     terminator      complement(838..881)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     regulatory      1005..1089
                     /regulatory_class="ribosome_binding_site"
     CDS             1087..1800
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Pedelacq et al., 2006)"
     CDS             1807..1830
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             1831..1854
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             1855..1878
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             1882..1899
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      1922..1949
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     CDS             complement(2181..3464)
                     /codon_start=1
                     /product="NBU integrase"
                     /label=NBU integrase
                     /protein_id="ARI71345.1"
                     /translation="MNIKRNIIFALESRKKNGVPIVENVPIRMRVIFASQRIEFTTGYR
                     IDVAKWDADKQRVKSGCTNKLKQSAAEINTDLLKYYAEIQNIFKEFEVQEVMPTTQQLK
                     EAFNMRMKDTSEEQPEEAPVSFWEVFDEFVKECGNQNNWTASTYEKFAAVRNHLKEFKE
                     DATFNYFNEFGLNEYVNFLRDTKDMRNSTIGKQMGFLKWFLRWSFKKGHHQNIAYDTFK
                     PKLKTTSKKVIFLTWDELNKLKDYQIPKDKQYLERVRDVFLFCCFTSLRYSDVRNLKRS
                     DVKSDHIEITTVKTADSLTIELNKYSKAILDKYKDIHFENYMALPVISNQKMNDYLKEL
                     GELAEINEPVRETYYKGNERIDEVTPKYALLSTHAGRRTFICNALALGIPAQVVMKWTG
                     HSDYKAMKPYIDIADDIKANAMNKFNQL"
     misc_feature    3624..3635
                     /label=integration site
                     /note="integration site"
     CDS             complement(3907..4764)
                     /label=AmpR
                     /note="beta-lactamase"
     CDS             5252..5986
                     /codon_start=1
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="ARI71348.1"
                     /translation="MNKVNIKDSQNFITSKYHIEKIMNCISLDEKDNIFEIGAGKGHFT
                     AGLVKRCNFVTAIEIDSKLCEVTRNKLLNYPNYQIVNDDILKFTFPSHNPYKIFGSIPY
                     NISTNIIRKIVFESSATISYLIVEYGFAKMLLDTNRSLALLLMAEVDISILAKIPRYYF
                     HPKPKVDSTLIVLKRKPAKMAFKERKKYETFVMKWVNKEYEKLFTKNQFNKALKHARIY
                     DINNISFEQFVSLFNSYKIFNG"
ORIGIN
        1 caccgcggtg gaggggaatt cccatgtcag ccgttaagtg ttcctgtgtc actcaaaatt
       61 gctttgagag gctctaaggg cttctcagtg cgttacatcc ctggcttgtt gtccacaacc
      121 gttaaacctt aaaagcttta aaagccttat atattctttt ttttcttata aaacttaaaa
      181 ccttagaggc tatttaagtt gctgatttat attaatttta ttgttcaaac atgagagctt
      241 agtacgtgaa acatgagagc ttagtacgtt agccatgaga gcttagtacg ttagccatga
      301 gggtttagtt cgttaaacat gagagcttag tacgttaaac atgagagctt agtacgtgaa
      361 acatgagagc ttagtacgta ctatcaacag gttgaactgc tgatcttcag atcctctacg
      421 ccggacgcat cgtggccgga tcaattccgt tttccgctgc ataaccctgc ttcggggtca
      481 ttatagcgat tttttcggta tatccatcct ttttcgcacg atatacagga ttttgccaaa
      541 gggttcgtgt agactttcct tggtgtatcc aacggcgtca gccgggcagg ataggtgaag
      601 taggcccacc cgcgagcggg tgttccttct tcactgtccc ttattcgcac ctggcggtgc
      661 tcaacgggaa tcctgctctg cgaggctggc cggctaccgc cggcgtaaca gatgagggca
      721 agcggatggc tgatgaaacc aagccaacca ggaagggcag cccacctatc acggaattga
      781 tccccctcga attggaattc gttccgtctc gattcagatc taaaggatcc aatctagata
      841 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta caattgggct accttttttt
      901 tgttttgttt gcaatggtta atctattgtt aaaatttaaa gtttcacttg aactttcaaa
      961 taatgttctt atatttgcag tgtcgaaaga aacaaagtag gactgatcac acaacattta
     1021 aaaaataaca ttatgaaagc actcgaaaag agattcaaag gtaatattaa caataattta
     1081 ttttcaatgc gtaaaggcga agagctgttc actggtgtcg tccctattct ggtggaactg
     1141 gatggtgatg tcaacggtca taagttttcc gtgcgtggcg agggtgaagg tgacgcaact
     1201 aatggtaaac tgacgctgaa gttcatctgt actactggta aactgccggt tccttggccg
     1261 actctggtaa cgacgctgac ttatggtgtt cagtgctttg ctcgttatcc ggaccatatg
     1321 aagcagcatg acttcttcaa gtccgccatg ccggaaggct atgtgcagga acgcacgatt
     1381 tcctttaagg atgacggcac gtacaaaacg cgtgcggaag tgaaatttga aggcgatacc
     1441 ctggtaaacc gcattgagct gaaaggcatt gactttaaag aagacggcaa tatcctgggc
     1501 cataagctgg aatacaattt taacagccac aatgtttaca tcaccgccga taaacaaaaa
     1561 aatggcatta aagcgaattt taaaattcgc cacaacgtgg aggatggcag cgtgcagctg
     1621 gctgatcact accagcaaaa cactccaatc ggtgatggtc ctgttctgct gccagacaat
     1681 cactatctga gcacgcaaag cgttctgtct aaagatccga acgagaaacg cgatcatatg
     1741 gttctgctgg agttcgtaac cgcagcgggc atcacgcatg gtatggatga actgtacaaa
     1801 ggatctgatt acaaggacga tgatgataag gactacaaag atgatgatga caaagattat
     1861 aaggacgatg atgataaagg tcatcatcat catcatcact aaatcctagc tgattagctc
     1921 gagaaggcca tcctgacgga tggccttttt tttgtatcga gacgactagt gtaattgcct
     1981 atcttccagt gatggaacag catttgtgca ttggctgcaa caatcagcct tacttgtgcc
     2041 tgttctattt ccgaaccgac cgcttgtatg aatccatcaa aattcgtttt ctctatgttg
     2101 gattccttgt tgctcatatt gtgatgataa tttctacaaa tatagtcatt ggtaactatc
     2161 tatgaaactg tttgatactt ttatagttga ttaaacttgt tcatggcatt tgccttaata
     2221 tcatccgcta tgtcaatgta gggtttcata gctttgtagt cgctgtgtcc cgtccatttc
     2281 atgaccacct gtgccgggat tccgagagcc agcgcattgc agatgaatgt ccttcttcct
     2341 gcatgggtac tgagcaaagc gtatttgggt gtgacttcat caatacgttc atttcccttg
     2401 tagtaggttt cccgtacagg ctcgttgatt tctgccagtt cgcccagctc tttcaggtaa
     2461 tcgttcatct tctggttgct gatgacgggc agagccatgt aattctcgaa atggatgtcc
     2521 ttgtatttgt ccagtatggc tttgctgtat ttgttcagtt caatcgtcag gctgtcggca
     2581 gtcttgactg tggttatttc gatgtggtcg gacttcacat cgcttctttt cagattgcga
     2641 acatccgaat accgcaaact cgtaaagcag cagaacagga aaacatcacg cacacgttcc
     2701 aggtattgct tatccttggg tatctggtag tctttcagct tgttcagttc atcccaagtc
     2761 aggaagatta cttttttcga ggtggttttc agtttcggtt tgaacgtatc gtatgcaatg
     2821 ttctgatgat gtcctttctt gaagctccag cgcaggaacc atttgaggaa tcccatttgc
     2881 ttgccgatgg tgctgtttct catatccttg gtgtcacgca ggaagttgac gtattcgttc
     2941 aatccaaact cgttgaaata gttgaacgtt gcatcctcct tgaactcttt gaggtggttc
     3001 ctcactgctg caaatttttc ataggtggat gccgtccagt tattctggtt accgcactct
     3061 tttacaaact catcgaacac ctcccaaaag ctgacagggg cttcttccgg ctgttcttcg
     3121 ctggtgtctt tcattctcat gttgaaagct tccttcaact gttgggtcgt tggcatgacc
     3181 tcctgcacct caaattcctt gaaaatattc tggatttcgg catagtattt cagcaagtcc
     3241 gtattgattt cggctgcact ttgctttagc ttgttggtac atccgctctt tacccgctgc
     3301 ttatctgcat cccatttggc tacgtcaatc cggtagcccg ttgtaaactc gatgcgttgg
     3361 ctggcaaaga tgacacgcat acggatgggt acgttctcta cgattggcac accgttcttt
     3421 ttccggctct ccaatgcaaa aatgatgttg cgcttgatat tcataattgg gtgcgtttga
     3481 aattctacac ccaaatatac acccaattat tgagatagca aaagacattt agaaacattt
     3541 acttttactc tatattgtaa tttacacttg attatcagtc gtttgcagtc ttatgatatt
     3601 ctgtgaaagt ataagttcga gagcctgtct ctccgcaaaa aacgctgaaa atcagcagat
     3661 tgcaaaacaa acaccctgtt ttacacccaa gaatgtaaag tcggctgttt ttgttttatt
     3721 taagataata caaccactac ataataaaag agtagcgata ttaaaagaat ccgatgagaa
     3781 aagactaata tttatctatc cattcagttt gatttttcag gactttacat cgtcctgaaa
     3841 gtatttgttg gtaccgagga cgcgacagct gcagacgcgt tatgagtaaa cttggtctga
     3901 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3961 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4021 ccccagtgct gcaatgatac cgcgggaccc acgctcaccg gctccagatt tatcagcaat
     4081 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4141 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4201 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4261 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4321 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4381 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4441 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4501 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4561 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4621 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4681 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4741 gacacggaaa tgttgaatac tcatactctt cctttttcaa tcatggccgc gggattaaaa
     4801 gtcggggatt ggtgaacaaa aaggtgtttc tctctttaag agaaatatcg ttttgctaaa
     4861 cagttgatat tgaggtatca ttttatcgta aaagacattt ttgctcaaca attgcttgac
     4921 ggaaatcaac aaattttagc attttgtaaa aaagtcgcta tataatttgg tgaattggag
     4981 ttattttcat atttttgcat cccgaagagt ttctcttaaa gagagaaaca tcttttgcat
     5041 accttttccg accgaatttt tatgtcgtaa agaggggctt tgcagggggt ggactcagaa
     5101 agatgagaat agatgactat tgtagttgaa acacatagaa agttgctgat atacagaccg
     5161 atacgcatat cgggatgaac catgagtacg ttcttttctc aaaaaacata aatattcgaa
     5221 aagagatgca ataaattaag gagaggttat aatgaacaaa gtaaatataa aagatagtca
     5281 aaattttatt acttcaaaat atcacataga aaaaataatg aattgcataa gtttagatga
     5341 aaaagataac atctttgaaa taggtgcagg gaaaggtcat tttactgctg gattggtaaa
     5401 gagatgtaat tttgtaacgg cgatagaaat tgattctaaa ttatgtgagg taactcgtaa
     5461 taagctctta aattatccta actatcaaat agtaaatgat gatatactga aatttacatt
     5521 tcctagccac aatccatata aaatatttgg cagcatacct tacaacataa gcacaaatat
     5581 aattcgaaaa attgtttttg aaagttcagc cacaataagt tatttaatag tggaatatgg
     5641 ttttgctaaa atgttattag atacaaacag atcactagca ttgctgttaa tggcagaggt
     5701 agatatttct atattagcaa aaattcctag gtattatttc catccaaaac ctaaagtgga
     5761 tagcacatta attgtattaa aaagaaagcc agcaaaaatg gcatttaaag agagaaaaaa
     5821 atatgaaact tttgtaatga aatgggttaa caaagagtac gaaaaactgt ttacaaaaaa
     5881 tcaatttaat aaagctttaa aacatgcgag aatatatgat ataaacaata ttagtttcga
     5941 acaatttgta tcgctattta atagttataa aatatttaac ggctaaaaac aataggccac
     6001 atgcaactgt aaatgtttac gcgggtaccg a
//
