LOCUS       40924_5229        7549 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Bacterial two-hybrid vector pB2H-delta-alfa, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7549)
  AUTHORS   De Schamphelaire W, Olbrechts A, Meert J, Verhelst K, Roggeman 
            Fonseca M, Vanhoucke M, Beyaert R.
  TITLE     BCCM/LMBP Plasmid collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7549)
  AUTHORS   De Schamphelaire W.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-FEB-2017) BCCM/LMBP, Universiteit Gent, 
            Technologiepark 927, 9052, BELGIUM
REFERENCE   3  (bases 1 to 7549)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7549)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (02-FEB-2017) BCCM/LMBP, Universiteit Gent, Technologiepark 927, 
            9052, BELGIUM"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7549
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /label=lac operator
                     /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)."
     RBS             42..64
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             complement(78..3023)
                     /codon_start=1
                     /label=lacZ
                     /note="beta-galactosidase"
                     /translation="ARTDRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVV
                     PSNWQMHGYDAPIYTNVTYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDG
                     VNSAFHLWCNGRWVGYGQDSRLPSEFDLSAFLRAGENRLAVMVLRWSDGSYLEDQDMWR
                     MSGIFRDVSLLHKPTTQISDFHVATRFNDDFSRAVLEAEVQMCGELRDYLRVTVSLWQG
                     ETQVASGTAPFGGEIIDERGGYADRVTLRLNVENPKLWSAEIPNLYRAVVELHTADGTL
                     IEAEACDVGFREVRIENGLLLLNGKPLLIRGVNRHEHHPLHGQVMDEQTMVQDILLMKQ
                     NNFNAVRCSHYPNHPLWYTLCDRYGLYVVDEANIETHGMVPMNRLTDDPRWLPAMSERV
                     TRMVQRDRNHPSVIIWSLGNESGHGANHDALYRWIKSVDPSRPVQYEGGGADTTATDII
                     CPMYARVDEDQPFPAVPKWSIKKWLSLPGETRPLILCEYAHAMGNSLGGFAKYWQAFRQ
                     YPRLQGGFVWDWVDQSLIKYDENGNPWSAYGGDFGDTPNDRQFCMNGLVFADRTPHPAL
                     TEAKHQQQFFQFRLSGQTIEVTSEYLFRHSDNELLHWMVALDGKPLASGEVPLDVAPQG
                     KQLIELPELPQPESAGQLWLTVRVVQPNATAWSEAGHISAWQQWRLAENLSVTLPAASH
                     AIPHLTTSEMDFCIELGNKRWQFNRQSGFLSQMWIGDKKQLLTPLRDQFTRAPLDNDIG
                     VSEATRIDPNAWVERWKAAGHYQAEAALLQCTADTLADAVLITTAHAWQHQGKTLFISR
                     KTYRIDGSGQMAITVDVEVASDTPHPARIGLNCQLAQVAERVNWLGLGPQENYPDRLTA
                     ACFDRWDLPLSDMYTPYVFPSENGLRCGTRELNYGPHQWRGDFQFNISRYSQQQLMETS
                     HRHLLHAEEGTWLNIDGFHMGIGGDDSWSPSVSAEFQLSAGRYHYQLVWCQK"
     misc_feature    complement(3118..3153)
                     /label=5' UTR of lacZ, incl. RBS
                     /note="5' UTR of lacZ, incl. RBS"
     protein_bind    3135..3151
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     promoter        complement(3159..3187)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     promoter        complement(3216..3234)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3252..3268)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3276..3292)
                     /label=lac operator
                     /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)."
     promoter        complement(3300..3330)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3345..3366)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        3683..3711
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     CDS             3907..3951
                     /codon_start=1
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /translation="KETAAAKFERQHMDS"
     terminator      4003..4050
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4273..4929)
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(4930..5032)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(5558..6102)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     CDS             complement(6330..7409)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7410..7487)
                     /label=lacI promoter
     promoter        7533..7549
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atggttattt ttgacaccag accaactggt aatggtagcg accggcgctc
      121 agctggaatt ccgccgatac tgacgggctc caggagtcgt cgccaccaat ccccatatgg
      181 aaaccgtcga tattcagcca tgtgccttct tccgcgtgca gcagatggcg atggctggtt
      241 tccatcagtt gctgttgact gtagcggctg atgttgaact ggaagtcgcc gcgccactgg
      301 tgtgggccat aattcaattc gcgcgtcccg cagcgcagac cgttttcgct cgggaagacg
      361 tacggggtat acatgtctga caatggcaga tcccagcggt caaaacaggc ggcagtaagg
      421 cggtcgggat agttttcttg cggccctaat ccgagccagt ttacccgctc tgctacctgc
      481 gccagctggc agttcaggcc aatccgcgcc ggatgcggtg tatcgctcgc cacttcaaca
      541 tcaacggtaa tcgccatttg accactacca tcaatccggt aggttttccg gctgataaat
      601 aaggttttcc cctgatgctg ccacgcgtga gcggtcgtaa tcagcaccgc atcagcaagt
      661 gtatctgccg tgcactgcaa caacgctgct tcggcctggt aatggcccgc cgccttccag
      721 cgttcgaccc aggcgttagg gtcaatgcgg gtcgcttcac ttacgccaat gtcgttatcc
      781 agcggtgcac gggtgaactg atcgcgcagc ggcgtcagca gttgtttttt atcgccaatc
      841 cacatctgtg aaagaaagcc tgactggcgg ttaaattgcc aacgcttatt acccagctcg
      901 atgcaaaaat ccatttcgct ggtggtcaga tgcgggatgg cgtgggacgc ggcggggagc
      961 gtcacactga ggttttccgc cagacgccac tgctgccagg cgctgatgtg cccggcttct
     1021 gaccatgcgg tcgcgttcgg ttgcactacg cgtactgtga gccagagttg cccggcgctc
     1081 tccggctgcg gtagttcagg cagttcaatc aactgtttac cttgtggagc gacatccaga
     1141 ggcacttcac cgcttgccag cggcttacca tccagcgcca ccatccagtg caggagctcg
     1201 ttatcgctat gacggaacag gtattcgctg gtcacttcga tggtttgccc ggataaacgg
     1261 aactggaaaa actgctgctg gtgttttgct tccgtcagcg ctggatgcgg cgtgcggtcg
     1321 gcaaagacca gaccgttcat acagaactgg cgatcgttcg gcgtatcgcc aaaatcaccg
     1381 ccgtaagccg accacgggtt gccgttttca tcatatttaa tcagcgactg atccacccag
     1441 tcccagacga agccgccctg taaacgggga tactgacgaa acgcctgcca gtatttagcg
     1501 aaaccgccaa gactgttacc catcgcgtgg gcgtattcgc aaaggatcag cgggcgcgtc
     1561 tctccaggta gcgaaagcca ttttttgatg gaccatttcg gcacagccgg gaagggctgg
     1621 tcttcatcca cgcgcgcgta catcgggcaa ataatatcgg tggccgtggt gtcggctccg
     1681 ccgccttcat actgcaccgg gcgggaagga tcgacagatt tgatccagcg atacagcgcg
     1741 tcgtgattag cgccgtggcc tgattcattc cccagcgacc agatgatcac actcgggtga
     1801 ttacgatcgc gctgcaccat tcgcgttacg cgttcgctca tcgccggtag ccagcgcgga
     1861 tcatcggtca gacgattcat tggcaccatg ccgtgggttt caatattggc ttcatccacc
     1921 acatacaggc cgtagcggtc gcacagcgtg taccacagcg gatggttcgg ataatgcgaa
     1981 cagcgcacgg cgttaaagtt gttctgcttc atcagcagga tatcctgcac catcgtctgc
     2041 tcatccatga cctgaccatg cagaggatga tgctcgtgac ggttaacgcc tcgaatcagc
     2101 aacggcttgc cgttcagcag cagcagacca ttttcaatcc gcacctcgcg gaaaccgaca
     2161 tcgcaggctt ctgcttcaat cagcgtgccg tcggcggtgt gcagttcaac caccgcacga
     2221 tagagattcg ggatttcggc gctccacagt ttcgggtttt cgacgttcag acgtagtgtg
     2281 acgcgatcgg cataaccacc acgctcatcg ataatttcac cgccgaaagg cgcggtgccg
     2341 ctggcgacct gcgtttcacc ctgccataaa gaaactgtta cccgtaggta gtcacgcaac
     2401 tcgccgcaca tctgaacttc agcctccagt acagcgcggc tgaaatcatc attaaagcga
     2461 gtggcaacat ggaaatcgct gatttgtgta gtcggtttat gcagcaacga gacgtcacgg
     2521 aaaatgccgc tcatccgcca catatcctga tcttccagat aactgccgtc actccaacgc
     2581 agcaccatca ccgcgaggcg gttttctccg gcgcgtaaaa atgcgctcag gtcaaattca
     2641 gacggcaaac gactgtcctg gccgtaaccg acccagcgcc cgttgcacca cagatgaaac
     2701 gccgagttaa cgccatcaaa aataattcgc gtctggcctt cctgtagcca gctttcatca
     2761 acattaaatg tgagcgagta acaacccgtc ggattctccg tgggaacaaa cggcggattg
     2821 accgtaatgg gataggttac gttggtgtag atgggcgcat cgtaaccgtg catctgccag
     2881 tttgagggga cgacgacagt atcggcctca ggaagatcgc actccagcca gctttccggc
     2941 accgcttctg gtgccggaaa ccaggcaaag cgccattcgc cattcaggct gcgcaactgt
     3001 tgggaagggc gatcggtgcg ggcaacgacg gccagtgaat ccgtaatcat ggtcataccc
     3061 ccggatccga attcgagctc ggcgcgcctg cagcggccgc atgcatatgg atcgatcctg
     3121 tttcctgtgt gaaattgtta tccgctcaca attccacaca ttatacgagc cgatgattaa
     3181 ttgtcaacag ggggatgggg agtaagcttg agtattctat agtgtcacct aaatagcttg
     3241 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     3301 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     3361 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3421 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     3481 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3541 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgaat
     3601 taattctcat gtttgacagc ttatcatcgg ataacccctt ggggcctcta aacgggtctt
     3661 gaggggtttt ttgttctcat gtttgacagc ttatcttcga ttagctttaa tgcggtagtt
     3721 tatcacagtt aaattgctaa cgcagtcagg caccgtgtat gaaatctaac aatgcgctca
     3781 tcgtcatcct cggcaccgtc accctggatg ctgtaggcat aggcttggtt atgccggtac
     3841 tgccgggcct cttgcgggat atcggccggc cacgcgatcg ctgacgtcgg taccctcgag
     3901 tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc
     3961 gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg
     4021 gcctctaaac gggtcttgag gggttttttg ctgaaacctc aggcatttga gaagcacacg
     4081 gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata agcggctatt
     4141 taacgaccct gccctgaacc gacgaccggg tcgaatttgc tttcgaattt ctgccattca
     4201 tccgcttatt atcacttatt caggcgtagc accaggcgtt taagggcacc aataactgcc
     4261 ttaaaaaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat
     4321 tctgccgaca tggaagccat cacagacggc atgatgaacc tgaatcgcca gcggcatcag
     4381 caccttgtcg ccttgcgtat aatatttgcc catagtgaaa acgggggcga agaagttgtc
     4441 catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg ctgagacgaa
     4501 aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac
     4561 atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga
     4621 tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat
     4681 caccagctca ccgtctttca ttgccatacg gaactccgga tgagcattca tcaggcgggc
     4741 aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa
     4801 ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc
     4861 ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt
     4921 tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg
     4981 tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat caacgtctca
     5041 ttttcgccaa aagttggccc agggcttccc ggtatcaaca gggacaccag gatttattta
     5101 ttctgcgaag tgatcttccg tcacaggtat ttattcggcg caaagtgcgt cgggtgatgc
     5161 tgccaactta ctgatttagt gtatgatggt gtttttgagg tgctccagtg gcttctgttt
     5221 ctatcagctg tccctcctgt tcagctactg acggggtggt gcgtaacggc aaaagcaccg
     5281 ccggacatca gcgctagcgg agtgtatact ggcttactat gttggcactg atgagggtgt
     5341 cagtgaagtg cttcatgtgg caggagaaaa aaggctgcac cggtgcgtca gcagaatatg
     5401 tgatacagga tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg
     5461 cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata
     5521 cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg
     5581 acaagcatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa
     5641 gataccaggc gtttcccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt
     5701 taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt
     5761 ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc
     5821 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac
     5881 cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt
     5941 taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg
     6001 ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg
     6061 ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat
     6121 cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
     6181 agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg
     6241 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     6301 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     6361 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     6421 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     6481 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     6541 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     6601 accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     6661 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     6721 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     6781 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     6841 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     6901 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     6961 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     7021 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     7081 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     7141 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     7201 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     7261 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     7321 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     7381 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     7441 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     7501 ctctccctta tgcgactcct gcattaggaa attaatacga ctcactata
//
