LOCUS       Exported                6987 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pWW3534, complete sequence.
ACCESSION   KY776516
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   Submitted (15-MAR-2017) Microbiology & Immunology, Stanford 
            University School of Medicine, 299 Campus Drive, Stanford, CA 
            94305-5124, USA
REFERENCE   3  (bases 1 to 6987)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6987
                     /organism="Cloning vector pWW3534"
                     /mol_type="other DNA"
                     /db_xref="taxon:1977342"
     regulatory      43..206
                     /regulatory_class="promoter"
     terminator      44..86
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     regulatory      211..295
                     /regulatory_class="ribosome_binding_site"
     CDS             293..1108
                     /codon_start=1
                     /product="green fluorescent protein - 3xFLAG 6xHis tag"
                     /label=green fluorescent protein - 3xFLAG 6xHis tag
                     /protein_id="ARI71356.1"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYKGSDYKDDDDKDYKDDDDKDYKDDDDKGHHHHHH"
     misc_feature    293..1006
                     /label=GFP domain of fusion protein
                     /note="GFP domain of fusion protein"
     CDS             293..1006
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     misc_feature    1007..1105
                     /label=epitope tag - 3xFLAG 6xHis
                     /note="epitope tag - 3xFLAG 6xHis"
     CDS             1013..1084
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             1088..1105
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1128..1155
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     regulatory      1130..1159
                     /regulatory_class="terminator"
     regulatory      1175..1267
                     /regulatory_class="promoter"
     regulatory      1272..1356
                     /regulatory_class="ribosome_binding_site"
     CDS             1354..2064
                     /codon_start=1
                     /product="mCherry"
                     /label=mCherry
                     /protein_id="ARI71358.1"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     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="ARI71354.1"
                     /translation="MNIKRNIIFALESRKKNGVPIVENVPIRMRVIFASQRIEFTTGYR
                     IDVAKWDADKQRVKSGCTNKLKQSAAEINTDLLKYYAEIQNIFKEFEVQEVMPTTQQLK
                     EAFNMRMKDTSEEQPEEAPVSFWEVFDEFVKECGNQNNWTASTYEKFAAVRNHLKEFKE
                     DATFNYFNEFGLNEYVNFLRDTKDMRNSTIGKQMGFLKWFLRWSFKKGHHQNIAYDTFK
                     PKLKTTSKKVIFLTWDELNKLKDYQIPKDKQYLERVRDVFLFCCFTSLRYSDVRNLKRS
                     DVKSDHIEITTVKTADSLTIELNKYSKAILDKYKDIHFENYMALPVISNQKMNDYLKEL
                     GELAEINEPVRETYYKGNERIDEVTPKYALLSTHAGRRTFICNALALGIPAQVVMKWTG
                     HSDYKAMKPYIDIADDIKANAMNKFNQL"
     misc_feature    3786..3797
                     /label=integration site
                     /note="integration site"
     misc_feature    4065..4662
                     /label=split antibiotic resistance marker
                     /note="split antibiotic resistance marker"
     CDS             complement(4066..4926)
                     /codon_start=1
                     /product="AmpR"
                     /label=AmpR
                     /protein_id="ARI71355.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     misc_feature    4659..4926
                     /label=split antibiotic resistance marker
                     /note="split antibiotic resistance marker"
     CDS             5414..6148
                     /codon_start=1
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="ARI71357.1"
                     /translation="MNKVNIKDSQNFITSKYHIEKIMNCISLDEKDNIFEIGAGKGHFT
                     AGLVKRCNFVTAIEIDSKLCEVTRNKLLNYPNYQIVNDDILKFTFPSHNPYKIFGSIPY
                     NISTNIIRKIVFESSATISYLIVEYGFAKMLLDTNRSLALLLMAEVDISILAKIPRYYF
                     HPKPKVDSTLIVLKRKPAKMAFKERKKYETFVMKWVNKEYEKLFTKNQFNKALKHARIY
                     DINNISFEQFVSLFNSYKIFNG"
     rep_origin      6214..6987
     rep_origin      6218..6606
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     oriT            6777..6886
                     /note="incP origin of transfer"