LOCUS       40924_46758        6325 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pWW3561, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6325)
  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 6325)
  AUTHORS   Whitaker WR, Shepherd ES, Sonnenburg JL.
  TITLE     Direct Submission
  JOURNAL   
REFERENCE   3  (bases 1 to 6325)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6325)
  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..6325
                     /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"
     terminator      1056..1127
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      1143..1170
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        1287..1337
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      1362..1384
                     /regulatory_class="ribosome_binding_site"
     CDS             1385..2098
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Pedelacq et al., 2006)"
     CDS             2105..2128
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             2129..2152
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             2153..2176
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             2180..2197
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      2220..2247
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     CDS             complement(2479..3762)
                     /codon_start=1
                     /product="NBU integrase"
                     /label=NBU integrase
                     /protein_id="ARI71368.1"
                     /translation="MNIKRNIIFALESRKKNGVPIVENVPIRMRVIFASQRIEFTTGYR
                     IDVAKWDADKQRVKSGCTNKLKQSAAEINTDLLKYYAEIQNIFKEFEVQEVMPTTQQLK
                     EAFNMRMKDTSEEQPEEAPVSFWEVFDEFVKECGNQNNWTASTYEKFAAVRNHLKEFKE
                     DATFNYFNEFGLNEYVNFLRDTKDMRNSTIGKQMGFLKWFLRWSFKKGHHQNIAYDTFK
                     PKLKTTSKKVIFLTWDELNKLKDYQIPKDKQYLERVRDVFLFCCFTSLRYSDVRNLKRS
                     DVKSDHIEITTVKTADSLTIELNKYSKAILDKYKDIHFENYMALPVISNQKMNDYLKEL
                     GELAEINEPVRETYYKGNERIDEVTPKYALLSTHAGRRTFICNALALGIPAQVVMKWTG
                     HSDYKAMKPYIDIADDIKANAMNKFNQL"
     misc_feature    3922..3933
                     /label=integration site
                     /note="integration site"
     CDS             complement(4205..5062)
                     /label=AmpR
                     /note="beta-lactamase"
     CDS             5550..6284
                     /codon_start=1
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="ARI71371.1"
                     /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 aaggacttat gccaacacaa ttaacatctc
      961 aatcaaggta aatgcttttt gctttttttg ctcggccggc ttgtcgacga cggccgtctc
     1021 cgtcgtcagg atcatccggg ccatatgcca ggcatcaaat aaaacgaaag gctcagtcga
     1081 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca
     1141 ctggctcacc ttcgggtggg cctttctgcg tttataatgc atgggtttgt accgtacacc
     1201 actgagacgg cggtggttga ccagacaaac cacgaaaagg agttttttag ttaccttaat
     1261 tgaaataaac gaaataaaaa ctcgcagctg tcaccggatg tgctttccgg tctgatgagt
     1321 ccgtgaggac gaaacagcct ctacaaataa ttttgtttaa cgggttatga aaaaatataa
     1381 gaaaatgcgt aaaggcgaag agctgttcac tggtgtcgtc cctattctgg tggaactgga
     1441 tggtgatgtc aacggtcata agttttccgt gcgtggcgag ggtgaaggtg acgcaactaa
     1501 tggtaaactg acgctgaagt tcatctgtac tactggtaaa ctgccggttc cttggccgac
     1561 tctggtaacg acgctgactt atggtgttca gtgctttgct cgttatccgg accatatgaa
     1621 gcagcatgac ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac gcacgatttc
     1681 ctttaaggat gacggcacgt acaaaacgcg tgcggaagtg aaatttgaag gcgataccct
     1741 ggtaaaccgc attgagctga aaggcattga ctttaaagaa gacggcaata tcctgggcca
     1801 taagctggaa tacaatttta acagccacaa tgtttacatc accgccgata aacaaaaaaa
     1861 tggcattaaa gcgaatttta aaattcgcca caacgtggag gatggcagcg tgcagctggc
     1921 tgatcactac cagcaaaaca ctccaatcgg tgatggtcct gttctgctgc cagacaatca
     1981 ctatctgagc acgcaaagcg ttctgtctaa agatccgaac gagaaacgcg atcatatggt
     2041 tctgctggag ttcgtaaccg cagcgggcat cacgcatggt atggatgaac tgtacaaagg
     2101 atctgattac aaggacgatg atgataagga ctacaaagat gatgatgaca aagattataa
     2161 ggacgatgat gataaaggtc atcatcatca tcatcactaa atcctagctg attagctcga
     2221 gaaggccatc ctgacggatg gccttttttt tgtatcgaga cgactagtgt aattgcctat
     2281 cttccagtga tggaacagca tttgtgcatt ggctgcaaca atcagcctta cttgtgcctg
     2341 ttctatttcc gaaccgaccg cttgtatgaa tccatcaaaa ttcgttttct ctatgttgga
     2401 ttccttgttg ctcatattgt gatgataatt tctacaaata tagtcattgg taactatcta
     2461 tgaaactgtt tgatactttt atagttgatt aaacttgttc atggcatttg ccttaatatc
     2521 atccgctatg tcaatgtagg gtttcatagc tttgtagtcg ctgtgtcccg tccatttcat
     2581 gaccacctgt gccgggattc cgagagccag cgcattgcag atgaatgtcc ttcttcctgc
     2641 atgggtactg agcaaagcgt atttgggtgt gacttcatca atacgttcat ttcccttgta
     2701 gtaggtttcc cgtacaggct cgttgatttc tgccagttcg cccagctctt tcaggtaatc
     2761 gttcatcttc tggttgctga tgacgggcag agccatgtaa ttctcgaaat ggatgtcctt
     2821 gtatttgtcc agtatggctt tgctgtattt gttcagttca atcgtcaggc tgtcggcagt
     2881 cttgactgtg gttatttcga tgtggtcgga cttcacatcg cttcttttca gattgcgaac
     2941 atccgaatac cgcaaactcg taaagcagca gaacaggaaa acatcacgca cacgttccag
     3001 gtattgctta tccttgggta tctggtagtc tttcagcttg ttcagttcat cccaagtcag
     3061 gaagattact tttttcgagg tggttttcag tttcggtttg aacgtatcgt atgcaatgtt
     3121 ctgatgatgt cctttcttga agctccagcg caggaaccat ttgaggaatc ccatttgctt
     3181 gccgatggtg ctgtttctca tatccttggt gtcacgcagg aagttgacgt attcgttcaa
     3241 tccaaactcg ttgaaatagt tgaacgttgc atcctccttg aactctttga ggtggttcct
     3301 cactgctgca aatttttcat aggtggatgc cgtccagtta ttctggttac cgcactcttt
     3361 tacaaactca tcgaacacct cccaaaagct gacaggggct tcttccggct gttcttcgct
     3421 ggtgtctttc attctcatgt tgaaagcttc cttcaactgt tgggtcgttg gcatgacctc
     3481 ctgcacctca aattccttga aaatattctg gatttcggca tagtatttca gcaagtccgt
     3541 attgatttcg gctgcacttt gctttagctt gttggtacat ccgctcttta cccgctgctt
     3601 atctgcatcc catttggcta cgtcaatccg gtagcccgtt gtaaactcga tgcgttggct
     3661 ggcaaagatg acacgcatac ggatgggtac gttctctacg attggcacac cgttcttttt
     3721 ccggctctcc aatgcaaaaa tgatgttgcg cttgatattc ataattgggt gcgtttgaaa
     3781 ttctacaccc aaatatacac ccaattattg agatagcaaa agacatttag aaacatttac
     3841 ttttactcta tattgtaatt tacacttgat tatcagtcgt ttgcagtctt atgatattct
     3901 gtgaaagtat aagttcgaga gcctgtctct ccgcaaaaaa cgctgaaaat cagcagattg
     3961 caaaacaaac accctgtttt acacccaaga atgtaaagtc ggctgttttt gttttattta
     4021 agataataca accactacat aataaaagag tagcgatatt aaaagaatcc gatgagaaaa
     4081 gactaatatt tatctatcca ttcagtttga tttttcagga ctttacatcg tcctgaaagt
     4141 atttgttggt accgaggacg cgacagctgc agacgcgtta tgagtaaact tggtctgaca
     4201 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4261 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4321 ccagtgctgc aatgataccg cgggacccac gctcaccggc tccagattta tcagcaataa
     4381 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4441 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4501 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4561 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4621 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4681 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4741 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4801 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4861 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4921 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4981 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5041 cacggaaatg ttgaatactc atactcttcc tttttcaatc atggccgcgg gattaaaagt
     5101 cggggattgg tgaacaaaaa ggtgtttctc tctttaagag aaatatcgtt ttgctaaaca
     5161 gttgatattg aggtatcatt ttatcgtaaa agacattttt gctcaacaat tgcttgacgg
     5221 aaatcaacaa attttagcat tttgtaaaaa agtcgctata taatttggtg aattggagtt
     5281 attttcatat ttttgcatcc cgaagagttt ctcttaaaga gagaaacatc ttttgcatac
     5341 cttttccgac cgaattttta tgtcgtaaag aggggctttg cagggggtgg actcagaaag
     5401 atgagaatag atgactattg tagttgaaac acatagaaag ttgctgatat acagaccgat
     5461 acgcatatcg ggatgaacca tgagtacgtt cttttctcaa aaaacataaa tattcgaaaa
     5521 gagatgcaat aaattaagga gaggttataa tgaacaaagt aaatataaaa gatagtcaaa
     5581 attttattac ttcaaaatat cacatagaaa aaataatgaa ttgcataagt ttagatgaaa
     5641 aagataacat ctttgaaata ggtgcaggga aaggtcattt tactgctgga ttggtaaaga
     5701 gatgtaattt tgtaacggcg atagaaattg attctaaatt atgtgaggta actcgtaata
     5761 agctcttaaa ttatcctaac tatcaaatag taaatgatga tatactgaaa tttacatttc
     5821 ctagccacaa tccatataaa atatttggca gcatacctta caacataagc acaaatataa
     5881 ttcgaaaaat tgtttttgaa agttcagcca caataagtta tttaatagtg gaatatggtt
     5941 ttgctaaaat gttattagat acaaacagat cactagcatt gctgttaatg gcagaggtag
     6001 atatttctat attagcaaaa attcctaggt attatttcca tccaaaacct aaagtggata
     6061 gcacattaat tgtattaaaa agaaagccag caaaaatggc atttaaagag agaaaaaaat
     6121 atgaaacttt tgtaatgaaa tgggttaaca aagagtacga aaaactgttt acaaaaaatc
     6181 aatttaataa agctttaaaa catgcgagaa tatatgatat aaacaatatt agtttcgaac
     6241 aatttgtatc gctatttaat agttataaaa tatttaacgg ctaaaaacaa taggccacat
     6301 gcaactgtaa atgtttacgc gggta
//
