LOCUS       Exported                6671 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector p4eng3VT-lacI, complete sequence.
ACCESSION   KX986159
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6671)
  AUTHORS   Boehm CR, Grant PK, Haseloff J.
  TITLE     Programmed emergence of a domain of gene expression in a bacterial 
            population
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6671)
  AUTHORS   Boehm CR.
  TITLE     Direct Submission
  JOURNAL   Submitted (12-OCT-2016) Plant Sciences, University of Cambridge, 
            Downing Street, Cambridge, Cambridgeshire CB2 3EA, United Kingdom
REFERENCE   3  (bases 1 to 6671)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..6671
                     /organism="Cloning vector p4eng3VT-lacI"
                     /mol_type="other DNA"
                     /db_xref="taxon:2049409"
     CDS             19..969
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     misc_feature    1187..1271
                     /note="PT7(-3G) and transl. enh. RBS from pET3a (Rosenberg 
                     et al. 1987)"
     RBS             1244..1266
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1278..1997
                     /codon_start=1
                     /product="mVenus"
                     /label=mVenus
                     /protein_id="ATP60595.1"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     regulatory      2009..2137
                     /regulatory_class="terminator"
                     /note="BBa_B0015"
     terminator      2017..2088
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2104..2131
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     regulatory      2138..2172
                     /regulatory_class="promoter"
                     /note="PJ23101*"
     misc_feature    2181..3488
                     /note="lacI_generator cassette; BBa_P0412"
     RBS             2181..2192
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2208..3287
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     CDS             3288..3320
                     /codon_start=1
                     /product="C-terminal peptide that mediates degradation in 
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
                     /label=ssrA tag (LVA)
                     /note="mutant LVA variant that confers accelerated 
                     degradation under some conditions (Andersen et al., 1998)"
                     /translation="AANDENYALVA"
     terminator      3368..3439
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3455..3482
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    3489..3509
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     CDS             3613..4473
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     terminator      4603..4632
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     misc_feature    4679..4700
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     regulatory      complement(4701..4829)
                     /regulatory_class="terminator"
                     /note="BBa_B0015"
     terminator      4707..4734
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      4750..4821
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     regulatory      complement(4841..5578)
                     /regulatory_class="ribosome_binding_site"
                     /note="BBa_B0034"
     CDS             complement(4841..5560)
                     /codon_start=1
                     /product="mTurquoise2"
                     /label=mTurquoise2
                     /protein_id="ATP60596.1"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     RBS             5567..5578
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     regulatory      complement(5587..5621)
                     /regulatory_class="promoter"
                     /note="pJ23101"
     misc_feature    5622..5642
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      5678..5709
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     terminator      5834..5928
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      6420..6642
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"