LOCUS       40924_46748        6987 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pWW3535, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6987)
  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 6987)
  AUTHORS   Whitaker WR, Shepherd ES, Sonnenburg JL.
  TITLE     Direct Submission
  JOURNAL   
REFERENCE   3  (bases 1 to 6987)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6987)
  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..6987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(44..87)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     regulatory      211..295
                     /regulatory_class="ribosome_binding_site"
     CDS             293..1006
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Pedelacq et al., 2006)"
     CDS             1013..1036
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             1037..1060
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             1061..1084
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             1088..1105
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      1128..1155
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     regulatory      1175..1267
                     /regulatory_class="promoter"
     regulatory      1272..1356
                     /regulatory_class="ribosome_binding_site"
     CDS             1354..2061
                     /label=mCherry
                     /note="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
     terminator      2084..2111
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     regulatory      2086..2115
                     /regulatory_class="terminator"
     CDS             complement(2343..3626)
                     /codon_start=1
                     /product="NBU integrase"
                     /label=NBU integrase
                     /protein_id="ARI71359.1"
                     /translation="MNIKRNIIFALESRKKNGVPIVENVPIRMRVIFASQRIEFTTGYR
                     IDVAKWDADKQRVKSGCTNKLKQSAAEINTDLLKYYAEIQNIFKEFEVQEVMPTTQQLK
                     EAFNMRMKDTSEEQPEEAPVSFWEVFDEFVKECGNQNNWTASTYEKFAAVRNHLKEFKE
                     DATFNYFNEFGLNEYVNFLRDTKDMRNSTIGKQMGFLKWFLRWSFKKGHHQNIAYDTFK
                     PKLKTTSKKVIFLTWDELNKLKDYQIPKDKQYLERVRDVFLFCCFTSLRYSDVRNLKRS
                     DVKSDHIEITTVKTADSLTIELNKYSKAILDKYKDIHFENYMALPVISNQKMNDYLKEL
                     GELAEINEPVRETYYKGNERIDEVTPKYALLSTHAGRRTFICNALALGIPAQVVMKWTG
                     HSDYKAMKPYIDIADDIKANAMNKFNQL"
     misc_feature    3786..3797
                     /label=integration site
                     /note="integration site"
     CDS             complement(4069..4926)
                     /label=AmpR
                     /note="beta-lactamase"
     CDS             5414..6148
                     /codon_start=1
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="ARI71362.1"
                     /translation="MNKVNIKDSQNFITSKYHIEKIMNCISLDEKDNIFEIGAGKGHFT
                     AGLVKRCNFVTAIEIDSKLCEVTRNKLLNYPNYQIVNDDILKFTFPSHNPYKIFGSIPY
                     NISTNIIRKIVFESSATISYLIVEYGFAKMLLDTNRSLALLLMAEVDISILAKIPRYYF
                     HPKPKVDSTLIVLKRKPAKMAFKERKKYETFVMKWVNKEYEKLFTKNQFNKALKHARIY
                     DINNISFEQFVSLFNSYKIFNG"
     rep_origin      complement(6218..6606)
                     /direction=LEFT
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     oriT            6777..6886
                     /label=oriT
                     /note="incP origin of transfer"
ORIGIN
        1 gaattcgttc cgtctcgatt cagatctaaa ggatccaatc tagataaaac gaaaggctca
       61 gtcgaaagac tgggcctttc gttttacaat tgggctacct tttttttgtt ttgtttgcaa
      121 tggttaatct attgttaaaa tttaaagttt cacttgaact ttcaaataat gttcttatat
      181 ttgcagtgtc gaaagaaaca aagtaggact gatcacacaa catttaaaaa ataacattat
      241 gaaagcactc gaaaagagat tcaaaggtaa tattaacaat aatttatttt caatgcgtaa
      301 aggcgaagag ctgttcactg gtgtcgtccc tattctggtg gaactggatg gtgatgtcaa
      361 cggtcataag ttttccgtgc gtggcgaggg tgaaggtgac gcaactaatg gtaaactgac
      421 gctgaagttc atctgtacta ctggtaaact gccggttcct tggccgactc tggtaacgac
      481 gctgacttat ggtgttcagt gctttgctcg ttatccggac catatgaagc agcatgactt
      541 cttcaagtcc gccatgccgg aaggctatgt gcaggaacgc acgatttcct ttaaggatga
      601 cggcacgtac aaaacgcgtg cggaagtgaa atttgaaggc gataccctgg taaaccgcat
      661 tgagctgaaa ggcattgact ttaaagaaga cggcaatatc ctgggccata agctggaata
      721 caattttaac agccacaatg tttacatcac cgccgataaa caaaaaaatg gcattaaagc
      781 gaattttaaa attcgccaca acgtggagga tggcagcgtg cagctggctg atcactacca
      841 gcaaaacact ccaatcggtg atggtcctgt tctgctgcca gacaatcact atctgagcac
      901 gcaaagcgtt ctgtctaaag atccgaacga gaaacgcgat catatggttc tgctggagtt
      961 cgtaaccgca gcgggcatca cgcatggtat ggatgaactg tacaaaggat ctgattacaa
     1021 ggacgatgat gataaggact acaaagatga tgatgacaaa gattataagg acgatgatga
     1081 taaaggtcat catcatcatc atcactaaat cctagctgat tagctcgaga aggccatcct
     1141 gacggatggc cttttttttg tatcgagacg actagagttt gcaatggtta atctattgtt
     1201 aaaatttaaa gtttcacttg aactttcaaa taatgttctt atatttgcag tgtcgaaaga
     1261 aacaaaggac tgatcacaca acatttaaaa aataacatta tgaaagcact cgaaaagaga
     1321 ttcaaaggta atattaacaa taatttattt tcaatggttt cgaaagggga agaagataac
     1381 atggctatca ttaaggagtt tatgcgcttc aaggtgcaca tggagggctc tgtcaacggc
     1441 cacgagttcg aaatcgaagg tgaaggtgaa ggtcgtccgt acgaaggtac tcagaccgca
     1501 aagttgaaag ttacaaaagg aggtccgttg cctttcgctt gggacattct gagcccgcaa
     1561 tttatgtacg ggagcaaagc ctatgttaaa catccggcgg atattcccga ttacctgaaa
     1621 ctgtcattcc cggaggggtt caagtgggaa cgcgtcatga actttgaaga cggtggcgtg
     1681 gttacagtga ctcaggattc gtcccttcaa gatggcgaat ttatctacaa agtcaaactg
     1741 cgcgggacaa attttccgag tgacggtcct gttatgcaga agaagaccat ggggtgggaa
     1801 gcaagctccg aacgcatgta ccccgaggac ggtgcactca aaggagaaat caagcagcgt
     1861 ctgaaactga aagatggcgg gcactacgat gcagaagtga aaaccaccta taaggcgaaa
     1921 aagcctgtac aactgccggg tgcctataat gtgaacatta aactggatat aacgagtcac
     1981 aacgaagact atacaatcgt cgagcagtat gagcgtgcgg aaggacgtca ttccactggt
     2041 ggaatggatg aactgtataa gtaaatccta gctgattagc tcgagaaggc catcctgacg
     2101 gatggccttt tttttgtatc gagacgacta gtgtaattgc ctatcttcca gtgatggaac
     2161 agcatttgtg cattggctgc aacaatcagc cttacttgtg cctgttctat ttccgaaccg
     2221 accgcttgta tgaatccatc aaaattcgtt ttctctatgt tggattcctt gttgctcata
     2281 ttgtgatgat aatttctaca aatatagtca ttggtaacta tctatgaaac tgtttgatac
     2341 ttttatagtt gattaaactt gttcatggca tttgccttaa tatcatccgc tatgtcaatg
     2401 tagggtttca tagctttgta gtcgctgtgt cccgtccatt tcatgaccac ctgtgccggg
     2461 attccgagag ccagcgcatt gcagatgaat gtccttcttc ctgcatgggt actgagcaaa
     2521 gcgtatttgg gtgtgacttc atcaatacgt tcatttccct tgtagtaggt ttcccgtaca
     2581 ggctcgttga tttctgccag ttcgcccagc tctttcaggt aatcgttcat cttctggttg
     2641 ctgatgacgg gcagagccat gtaattctcg aaatggatgt ccttgtattt gtccagtatg
     2701 gctttgctgt atttgttcag ttcaatcgtc aggctgtcgg cagtcttgac tgtggttatt
     2761 tcgatgtggt cggacttcac atcgcttctt ttcagattgc gaacatccga ataccgcaaa
     2821 ctcgtaaagc agcagaacag gaaaacatca cgcacacgtt ccaggtattg cttatccttg
     2881 ggtatctggt agtctttcag cttgttcagt tcatcccaag tcaggaagat tacttttttc
     2941 gaggtggttt tcagtttcgg tttgaacgta tcgtatgcaa tgttctgatg atgtcctttc
     3001 ttgaagctcc agcgcaggaa ccatttgagg aatcccattt gcttgccgat ggtgctgttt
     3061 ctcatatcct tggtgtcacg caggaagttg acgtattcgt tcaatccaaa ctcgttgaaa
     3121 tagttgaacg ttgcatcctc cttgaactct ttgaggtggt tcctcactgc tgcaaatttt
     3181 tcataggtgg atgccgtcca gttattctgg ttaccgcact cttttacaaa ctcatcgaac
     3241 acctcccaaa agctgacagg ggcttcttcc ggctgttctt cgctggtgtc tttcattctc
     3301 atgttgaaag cttccttcaa ctgttgggtc gttggcatga cctcctgcac ctcaaattcc
     3361 ttgaaaatat tctggatttc ggcatagtat ttcagcaagt ccgtattgat ttcggctgca
     3421 ctttgcttta gcttgttggt acatccgctc tttacccgct gcttatctgc atcccatttg
     3481 gctacgtcaa tccggtagcc cgttgtaaac tcgatgcgtt ggctggcaaa gatgacacgc
     3541 atacggatgg gtacgttctc tacgattggc acaccgttct ttttccggct ctccaatgca
     3601 aaaatgatgt tgcgcttgat attcataatt gggtgcgttt gaaattctac acccaaatat
     3661 acacccaatt attgagatag caaaagacat ttagaaacat ttacttttac tctatattgt
     3721 aatttacact tgattatcag tcgtttgcag tcttatgata ttctgtgaaa gtataagttc
     3781 gagagcctgt ctctccgcaa aaaacgctga aaatcagcag attgcaaaac aaacaccctg
     3841 ttttacaccc aagaatgtaa agtcggctgt ttttgtttta tttaagataa tacaaccact
     3901 acataataaa agagtagcga tattaaaaga atccgatgag aaaagactaa tatttatcta
     3961 tccattcagt ttgatttttc aggactttac atcgtcctga aagtatttgt tggtaccgag
     4021 gacgcgacag ctgcagacgc gttatgagta aacttggtct gacagttacc aatgcttaat
     4081 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4141 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4201 accgcgggac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4261 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4321 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4381 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4441 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4501 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4561 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4621 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4681 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4741 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4801 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4861 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4921 actcatactc ttcctttttc aatcatggcc gcgggattaa aagtcgggga ttggtgaaca
     4981 aaaaggtgtt tctctcttta agagaaatat cgttttgcta aacagttgat attgaggtat
     5041 cattttatcg taaaagacat ttttgctcaa caattgcttg acggaaatca acaaatttta
     5101 gcattttgta aaaaagtcgc tatataattt ggtgaattgg agttattttc atatttttgc
     5161 atcccgaaga gtttctctta aagagagaaa catcttttgc ataccttttc cgaccgaatt
     5221 tttatgtcgt aaagaggggc tttgcagggg gtggactcag aaagatgaga atagatgact
     5281 attgtagttg aaacacatag aaagttgctg atatacagac cgatacgcat atcgggatga
     5341 accatgagta cgttcttttc tcaaaaaaca taaatattcg aaaagagatg caataaatta
     5401 aggagaggtt ataatgaaca aagtaaatat aaaagatagt caaaatttta ttacttcaaa
     5461 atatcacata gaaaaaataa tgaattgcat aagtttagat gaaaaagata acatctttga
     5521 aataggtgca gggaaaggtc attttactgc tggattggta aagagatgta attttgtaac
     5581 ggcgatagaa attgattcta aattatgtga ggtaactcgt aataagctct taaattatcc
     5641 taactatcaa atagtaaatg atgatatact gaaatttaca tttcctagcc acaatccata
     5701 taaaatattt ggcagcatac cttacaacat aagcacaaat ataattcgaa aaattgtttt
     5761 tgaaagttca gccacaataa gttatttaat agtggaatat ggttttgcta aaatgttatt
     5821 agatacaaac agatcactag cattgctgtt aatggcagag gtagatattt ctatattagc
     5881 aaaaattcct aggtattatt tccatccaaa acctaaagtg gatagcacat taattgtatt
     5941 aaaaagaaag ccagcaaaaa tggcatttaa agagagaaaa aaatatgaaa cttttgtaat
     6001 gaaatgggtt aacaaagagt acgaaaaact gtttacaaaa aatcaattta ataaagcttt
     6061 aaaacatgcg agaatatatg atataaacaa tattagtttc gaacaatttg tatcgctatt
     6121 taatagttat aaaatattta acggctaaaa acaataggcc acatgcaact gtaaatgttt
     6181 acgcgggtac cgacaccgcg gtggagggga attcccatgt cagccgttaa gtgttcctgt
     6241 gtcactcaaa attgctttga gaggctctaa gggcttctca gtgcgttaca tccctggctt
     6301 gttgtccaca accgttaaac cttaaaagct ttaaaagcct tatatattct tttttttctt
     6361 ataaaactta aaaccttaga ggctatttaa gttgctgatt tatattaatt ttattgttca
     6421 aacatgagag cttagtacgt gaaacatgag agcttagtac gttagccatg agagcttagt
     6481 acgttagcca tgagggttta gttcgttaaa catgagagct tagtacgtta aacatgagag
     6541 cttagtacgt gaaacatgag agcttagtac gtactatcaa caggttgaac tgctgatctt
     6601 cagatcctct acgccggacg catcgtggcc ggatcaattc cgttttccgc tgcataaccc
     6661 tgcttcgggg tcattatagc gattttttcg gtatatccat cctttttcgc acgatataca
     6721 ggattttgcc aaagggttcg tgtagacttt ccttggtgta tccaacggcg tcagccgggc
     6781 aggataggtg aagtaggccc acccgcgagc gggtgttcct tcttcactgt cccttattcg
     6841 cacctggcgg tgctcaacgg gaatcctgct ctgcgaggct ggccggctac cgccggcgta
     6901 acagatgagg gcaagcggat ggctgatgaa accaagccaa ccaggaaggg cagcccacct
     6961 atcacggaat tgatccccct cgaattg
//
