LOCUS       Exported                4825 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  BioBrick cloning vector pSB4T5-I52001, complete sequence.
ACCESSION   EU496101
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4825)
  AUTHORS   Shetty RP, Endy D, Knight TF Jr.
  TITLE     Engineering BioBrick vectors from BioBrick parts
  JOURNAL   J Biol Eng 2, 5 (2008)
  PUBMED    18410688
REFERENCE   2  (bases 1 to 4825)
  AUTHORS   Shetty RP, Endy D, Knight TF Jr.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-FEB-2008) Biological Engineering, MIT, 32 Vassar 
            Street Room 32-311, Cambridge, MA 02139, USA
REFERENCE   3  (bases 1 to 4825)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4825
                     /organism="BioBrick cloning vector pSB4T5-I52001"
                     /mol_type="other DNA"
                     /db_xref="taxon:508437"
     misc_feature    complement(1..21)
                     /label=BioBrick cloning site suffix (BBa_G00001)
                     /note="BioBrick cloning site suffix (BBa_G00001)"
     misc_feature    35..46
                     /note="translational stop sequence for BioBrick 
                     vector-insert insulation, has stop codons in all 6 reading 
                     frames (BBa_B0042)"
     regulatory      47..115
                     /regulatory_class="terminator"
                     /note="derived from tonB terminator (BBa_B0054); similar to
                     Escherichia coli strain MG1655 accession U00096"
     stem_loop       57..88
                     /note="derived from tonB terminator, predicted using mFold"
     terminator      57..88
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     primer_bind     complement(116..135)
                     /note="VR reverse sequencing primer annealing site 
                     (BBa_G00102)"
     regulatory      136..176
                     /regulatory_class="terminator"
                     /note="derived from ribosomal RNA rrnC operon, reverse 
                     orientation (BBa_B0062); similar to Escherichia coli strain
                     MG1655 accession U00096"
     stem_loop       complement(149..168)
                     /label=derived from ribosomal RNA rrnC operon
                     /note="derived from ribosomal RNA rrnC operon"
     rep_origin      184..2220
                     /label=pSC101 low copy replication origin (BBa_I50042)
                     /note="pSC101 low copy replication origin (BBa_I50042)"
     terminator      213..307
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     misc_feature    320..687
                     /label=par region
                     /note="par region"
     rep_origin      799..1021
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     misc_feature    818..900
                     /label=AT rich region
                     /note="AT rich region"
     gene            1011..2019
                     /gene="rep101"
                     /label=rep101
     regulatory      1011..1016
                     /regulatory_class="minus_35_signal"
                     /gene="rep101"
     regulatory      1033..1038
                     /regulatory_class="minus_10_signal"
                     /gene="rep101"
     regulatory      1053..1065
                     /regulatory_class="ribosome_binding_site"
                     /gene="rep101"
     regulatory      1062..1067
                     /regulatory_class="ribosome_binding_site"
                     /gene="rep101"
     CDS             1069..2019
                     /codon_start=1
                     /gene="rep101"
                     /product="Rep101"
                     /label=rep101
                     /note="involved in plasmid replication"
                     /protein_id="ACB05495.1"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     misc_feature    2229..3511
                     /label=tet tetracycline resistance marker (BBa_P1005)
                     /note="tet tetracycline resistance marker (BBa_P1005)"
     promoter        2242..2270
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             2318..3508
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline resistance protein"
                     /label=tet
                     /note="confers resistance to tetracycline"
                     /protein_id="ACB05494.1"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     gene            2318..3508
                     /gene="tet"
                     /label=tet
     misc_feature    3396..3811
                     /label=ccdB positive selection marker (BBa_P1016)
                     /note="ccdB positive selection marker (BBa_P1016)"
     regulatory      3519..3590
                     /regulatory_class="terminator"
                     /note="derived from hisI terminator (BBa_B0053); similar to
                     Escherichia coli strain MG1655 accession U00096"
     stem_loop       3527..3561
                     /label=derived from hisI terminator
                     /note="derived from hisI terminator"
     primer_bind     3591..3610
                     /note="VF2 forward sequencing primer annealing site 
                     (BBa_G00100)"
     regulatory      3611..3688
                     /regulatory_class="terminator"
                     /note="derived from bacteriophage T3 terminator, reverse 
                     orientation (BBa_B0055); similar to bacteriophage T3 gene 1
                     to gene 11 (accession number X17255)"
     terminator      3638..3667
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     stem_loop       complement(3646..3667)
                     /label=derived from bacteriophage T3 terminator
                     /note="derived from bacteriophage T3 terminator"
     misc_feature    3689..3700
                     /note="translational stop sequence for BioBrick 
                     vector-insert insulation, has stop codons in all 6 reading 
                     frames (BBa_B0042)"
     misc_feature    3714..3735
                     /label=BioBrick cloning site prefix (BBa_G00000)
                     /note="BioBrick cloning site prefix (BBa_G00000)"
     gene            3776..4148
                     /gene="ccdB"
                     /label=ccdB
     regulatory      3776..3781
                     /regulatory_class="minus_35_signal"
                     /gene="ccdB"
     regulatory      3799..3804
                     /regulatory_class="minus_10_signal"
                     /gene="ccdB"
     CDS             3843..4148
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB"
                     /label=ccdB
                     /note="controlled cell death protein B; toxin protein that 
                     inhibits DNA gyrase which functions during DNA replication 
                     to catalyze negative supercoiling"
                     /protein_id="ACB05493.1"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     misc_feature    4152..4825
                     /note="nonfunctional high copy replication origin derived 
                     from pUC19 accession M77789 (BBa_I50020)"
     gene            4178..4766
                     /gene="RNAII"
                     /label=RNAII
     rep_origin      4178..4766
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     regulatory      4178..4183
                     /regulatory_class="minus_35_signal"
                     /gene="RNAII"
     regulatory      4199..4204
                     /regulatory_class="minus_10_signal"
                     /gene="RNAII"
     misc_RNA        4214..4766
                     /gene="RNAII"
                     /product="RNAII"
     gene            complement(4217..4360)
                     /gene="RNAI"
                     /label=RNAI
     misc_RNA        complement(4217..4324)
                     /gene="RNAI"
                     /product="RNAI"
     regulatory      complement(4333..4338)
                     /regulatory_class="minus_10_signal"
                     /gene="RNAI"
     regulatory      complement(4355..4360)
                     /regulatory_class="minus_35_signal"
                     /gene="RNAI"
     misc_difference 4412
                     /gene="RNAII"
                     /replace="g"
                     /note="G to A mutation to eliminate TspRI endonuclease 
                     site, renders BBa_I50020 nonfunctional as a replication 
                     origin"
     misc_difference 4416
                     /gene="RNAII"
                     /replace="g"
                     /note="G to A mutation to eliminate EcoP15I endonuclease 
                     site, renders BBa_I50020 nonfunctional as a replication 
                     origin"
     misc_difference 4425
                     /gene="RNAII"
                     /replace="g"
                     /note="G to A mutation to eliminate TspRI endonuclease 
                     site, renders BBa_I50020 nonfunctional as a replication 
                     origin"
     misc_difference 4454
                     /gene="RNAII"
                     /replace="c"
                     /note="C to T mutation to eliminate Nt.BstNBI endonuclease 
                     site, renders BBa_I50020 nonfunctional as a replication 
                     origin"
     misc_difference 4618
                     /gene="RNAII"
                     /replace="c"
                     /note="C to T mutation to eliminate BciVI endonuclease 
                     site, renders BBa_I50020 nonfunctional as a replication 
                     origin"