LOCUS       Exported                5437 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pKD233.7-3, complete sequence.
ACCESSION   MG323876
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5437)
  AUTHORS   Landry BP, Palanki R, Dyulgyarov N, Hartsough LA, Tabor JJ.
  TITLE     Phosphatase activity tunes two-component system sensor detection 
            threshold
  JOURNAL   Nat Commun 9 (1), 1433 (2018)
  PUBMED    29650958
REFERENCE   2  (bases 1 to 5437)
  AUTHORS   Landry BP.
  TITLE     Direct Submission
  JOURNAL   Submitted (29-OCT-2017) Bioengineering, Rice University, 6100 Main 
            St, Houston, TX 77005, USA
REFERENCE   3  (bases 1 to 5437)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5437
                     /organism="Cloning vector pKD233.7-3"
                     /mol_type="other DNA"
                     /db_xref="taxon:2219086"
     misc_feature    complement(1..6)
                     /label=AvrII Scar
                     /note="AvrII Scar"
     misc_feature    complement(6..819)
                     /label=ColE1 Fragment
                     /note="ColE1 Fragment"
     rep_origin      complement(133..815)
                     /direction=LEFT
                     /label=ColE1 origin
                     /note="ColE1 origin"
     rep_origin      complement(151..739)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(151..703)
                     /label=RNA II
                     /note="RNA II"
     regulatory      557..562
                     /regulatory_class="minus_35_signal"
     regulatory      580..586
                     /regulatory_class="minus_10_signal"
     misc_feature    593..700
                     /label=RNAI
                     /note="RNAI"
     regulatory      complement(711..717)
                     /regulatory_class="minus_10_signal"
     regulatory      complement(735..740)
                     /regulatory_class="minus_35_signal"
     misc_feature    complement(763..815)
                     /label=Tn3 Artifact
                     /note="Tn3 Artifact"
     misc_feature    complement(815..820)
                     /label=SpeI Scar
                     /note="SpeI Scar"
     regulatory      complement(820..925)
                     /regulatory_class="terminator"
                     /note="T0"
     terminator      827..921
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     misc_feature    complement(927..1721)
                     /label=CamR Cassette from E. coli R6-5 plasmid
                     /note="CamR Cassette from E. coli R6-5 plasmid"
     misc_feature    complement(927..932)
                     /label=SacI Scar
                     /note="SacI Scar"
     CDS             complement(942..1601)
                     /codon_start=1
                     /product="CmR"
                     /label=CmR
                     /protein_id="AWU50972.1"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(1602..1704)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     regulatory      1730..1764
                     /regulatory_class="promoter"
                     /note="constitutive promoter J23114"
     regulatory      1772..1785
                     /regulatory_class="ribosome_binding_site"
                     /note="RBS pprotet"
     regulatory      1774..1785
                     /regulatory_class="ribosome_binding_site"
                     /note="B0034"
     RBS             1774..1785
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1793..2503
                     /codon_start=1
                     /product="mCherry (RBS 7.923)"
                     /label=mCherry
                     /protein_id="AWU50970.1"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     misc_feature    2504..2507
                     /label=Scar
                     /note="Scar"
     regulatory      2508..2559
                     /regulatory_class="terminator"
                     /note="L3S1P52"
     misc_feature    2560..2563
                     /label=Scar
                     /note="Scar"
     misc_feature    2585..2635
                     /label=E. coli LacZ Upstream
                     /note="E. coli LacZ Upstream"
     misc_feature    2637..2642
                     /label=XhoI Scar
                     /note="XhoI Scar"
     promoter        2643..2716
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     regulatory      2643..2696
                     /regulatory_class="promoter"
                     /note="PLTetO-1"
     misc_feature    2643..2661
                     /label=TetR1
                     /note="TetR1"
     protein_bind    2643..2661
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      2662..2667
                     /regulatory_class="minus_35_signal"
     misc_feature    2668..2686
                     /label=TetR2
                     /note="TetR2"
     protein_bind    2668..2686
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      2685..2690
                     /regulatory_class="minus_10_signal"
     misc_feature    2697..2716
                     /label=Lambda Phage Fragment
                     /note="Lambda Phage Fragment"
     misc_feature    2697
                     /label=+1
                     /note="+1"
     regulatory      2717..2751
                     /regulatory_class="ribosome_binding_site"
                     /note="sRBS (1087.31)"
     CDS             2752..3372
                     /codon_start=1
                     /product="Sbal195_3858 (TtrR)"
                     /label=Sbal195_3858
                     /protein_id="AWU50974.1"
                     /translation="MSLALPVYLIDDDDSVRRSLRFMLESYGLKITDFDSAEAFFTAVD
                     LTLPGCALVDVRMPGLSGPQLHLELVSKNSPLAVIYLTGHGDVPMAVEALKLGAVDFFQ
                     KPADGAKLAEAVVKALEHAKTHYQDNQYLETYQALTPREREILNLIAQGLKNQEIADNL
                     CIAMRTVEIHRANLMKGMQVGSLAEMMLIYSRIAERLRLEDNR"
     misc_feature    2914..2916
                     /label=D55
                     /note="D55"
     regulatory      3382..3510
                     /regulatory_class="terminator"
                     /note="B0015"
     regulatory      3382..3461
                     /regulatory_class="terminator"
                     /note="B0010"
     terminator      3390..3461
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    3462..3469
                     /label=BB Scar
                     /note="BB Scar"
     regulatory      3470..3510
                     /regulatory_class="terminator"
                     /note="B0012"
     terminator      3477..3504
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    3511..3615
                     /label=Ptrp Fragment
                     /note="Ptrp Fragment"
     regulatory      3628..3712
                     /regulatory_class="promoter"
                     /note="Psbal195_3860 (185-269)"
     misc_feature    3630..3653
                     /label=Actual repeat
                     /note="Actual repeat"
     misc_feature    3630..3634
                     /label=Repeat
                     /note="Repeat"
     misc_feature    3635..3648
                     /label=Fnr
                     /note="Fnr"
     misc_feature    3642..3653
                     /label=Repeat
                     /note="Repeat"
     misc_feature    complement(3646..3650)
                     /label=Repeat
                     /note="Repeat"
     regulatory      3650..3655
                     /regulatory_class="minus_35_signal"
     regulatory      3676..3681
                     /regulatory_class="minus_10_signal"
     misc_feature    3688
                     /label=+1
                     /note="+1"
     misc_feature    3697..3703
                     /label=Repeat
                     /note="Repeat"
     misc_feature    complement(3709..3715)
                     /label=Repeat
                     /note="Repeat"
     regulatory      3713..3730
                     /regulatory_class="ribosome_binding_site"
                     /note="BBa_B0034"
     RBS             3713..3724
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3731..4447
                     /codon_start=1
                     /product="superfolder GFP"
                     /label=superfolder GFP
                     /protein_id="AWU50971.1"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             3731..4444
                     /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"
     regulatory      4463..4591
                     /regulatory_class="terminator"
                     /note="B0015"
     regulatory      4463..4542
                     /regulatory_class="terminator"
                     /note="B0010"
     terminator      4471..4542
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    4543..4550
                     /label=BB Scar
                     /note="BB Scar"
     regulatory      4551..4591
                     /regulatory_class="terminator"
                     /note="B0012"
     terminator      4558..4585
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    4592..4636
                     /label=pUC MCS
                     /note="pUC MCS"
     regulatory      4637..4671
                     /regulatory_class="promoter"
                     /note="constitutive promoter J23106"
     regulatory      4672..4705
                     /regulatory_class="ribosome_binding_site"
                     /note="sRBS (50,000)"
     CDS             4706..5332
                     /codon_start=1
                     /product="TetR"
                     /label=TetR
                     /note="tetR"
                     /protein_id="AWU50973.1"
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     regulatory      5333..5437
                     /regulatory_class="terminator"
                     /note="T1"
     terminator      5338..5424
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"