LOCUS       40924_39788        6886 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pSFFLAG-MAC, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6886)
  AUTHORS   Couch RD, Seidle HF, Parry RJ.
  TITLE     Construction of Expression Vectors to Produce Affinity Tagged 
            Proteins in Pseudomonas
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6886)
  AUTHORS   Couch RD, Seidle HF, Parry RJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (19-DEC-2001) Chemistry, Rice University, 1900 Rice Blvd 
            MS60, Houston, TX 77005, USA
REFERENCE   3  (bases 1 to 6886)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6886)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (19-DEC-2001) Chemistry, Rice University, 1900 Rice Blvd MS60, 
            Houston, TX 77005, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6886
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        39..67
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    75..91
                     /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)."
     CDS             115..138
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     terminator      377..463
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      555..582
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        601..692
                     /label=AmpR promoter
     CDS             693..1550
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRHDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1724..2312
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      complement(2924..3275)
                     /direction=LEFT
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     CDS             3289..4119
                     /codon_start=1
                     /label=pRO1600 Rep
                     /note="replication protein for the broad-host-range plasmid
                     pRO1600 from Pseudomonas aeruginosa"
                     /translation="VASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     primer_bind     4283..4299
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      4370..4821
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(4923..5096)
                     /codon_start=1
                     /label=lacZ-alpha
                     /note="LacZ-alpha fragment of beta-galactosidase"
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     protein_bind    complement(5116..5132)
                     /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(5140..5170)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5185..5206)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5222..6301)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6302..6379)
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
ORIGIN
        1 catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat catcggctcg
       61 tataatgtgt ggaattgtga gcggataaca atttcacaca ggagatatct tatggactac
      121 aaggacgacg atgacaaagt caagcttctc gagaattccc gggtacctgc agatctagat
      181 agatgagctc gtcgagtgag agaagatttt cagcctgata cagattaaat cagaagcggt
      241 ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
      301 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta
      361 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
      421 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt
      481 gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag
      541 gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct
      601 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
      661 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
      721 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
      781 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
      841 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
      901 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
      961 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1021 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1081 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1141 tgcacaacat gggggatcat gtaactcgcc atgatcgttg ggaaccggag ctgaatgaag
     1201 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     1261 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     1321 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     1381 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     1441 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     1501 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     1561 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     1621 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     1681 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     1741 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     1801 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     1861 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     1921 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     1981 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2041 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2101 acctacagcg tgagcattga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2161 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2221 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2281 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     2341 tcctggcctt ttgctggcct tttgctcaca tgtccacttc tgcgctcggc ccttccggct
     2401 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gatctcgcgg tatcattgca
     2461 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     2521 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     2581 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     2641 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     2701 cgtgagtttt cgttccactg agcgtcagac cccaattaca cgccactggc tgtgcttgct
     2761 ggggtgacgg tggcaacggt ggcggccttg ctgggctatc gcgttggaaa gaaacgaggg
     2821 aaaggggact gataaaccgg tcttagcccc tccccttggt gtccaaccgc tctgtaggcc
     2881 tctcaggcgc cgctggtgcc gctggttgga cgccaagggt gaatccgcct cgataccctg
     2941 attactcgct tcctgcgccc tctcaggcgg cgatagggga ctggtaaaac ggggattgcc
     3001 cagacgcctc ccccgcccct tcaggggcac aaatgcggcc ccaacggggc cacgtagtgg
     3061 tgcgtttttt gcgtttccac ccttttcttc cttttccctt ttaaaccttt taggacgtct
     3121 acaggccacg taatccgtgg cctgtagagt ttaaaaaggg acggatttgt tgccattaag
     3181 ggacggattt gttgttaaga agggacggat ttgttgttgt aaagggacgg atttgttgta
     3241 ttgtgggacg cagatacagt gtccccttat acacaaggaa tgtcgaacgt ggcctcaccc
     3301 ccaatggttt acaaaagcaa tgccctggtc gaggccgcgt atcgcctcag tgttcaggaa
     3361 cagcggatcg ttctggcctg tattagccag gtgaagagga gcgagcctgt caccgatgaa
     3421 gtgatgtatt cagtgacggc ggaggacata gcgacgatgg cgggtgtccc tatcgaatct
     3481 tcctacaacc agctcaaaga agcggccctg cgcctgaaac ggcgggaagt ccggttaacc
     3541 caagagccca atggcaaggg gaaaagaccg agtgtgatga ttaccggctg ggtgcaaaca
     3601 atcatctacc gggagggtga gggccgtgta gaactcaggt tcaccaaaga catgctgccg
     3661 tacctgacgg aactcaccaa acagttcacc aaatacgcct tggctgacgt ggccaagatg
     3721 gacagcaccc acgcgatcag gctttacgag ctgctcatgc aatgggacag catcggccag
     3781 cgcgaaatag aaattgacca gctgcgaaag tggtttcaac tggaaggccg gtatccctcg
     3841 atcaaggact tcaagttgcg agtgcttgat ccagccgtga cgcagatcaa cgagcacagc
     3901 ccgctacagg tggagtgggc gcagcgaaag accgggcgca aggtcacaca tctgttgttc
     3961 agttttggac cgaagaagcc cgccaaggcg gtgggtaagg ccccagcgaa gcgcaaggcc
     4021 gggaagattt cagatgctga gatcgcgaaa caggctcgcc ctggtgagac atgggaagcg
     4081 gcccgcgctc gactaaccca gatgccgctg gatctggcct agaggccgtg gccaccacgg
     4141 cccggcctgc ctttcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     4201 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
     4261 gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcggaagag cgcctgatgc
     4321 ggtattttct ccttacgcat ctgtgcggta tttcacaccg cagatcctga cgcgccctgt
     4381 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4441 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4501 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4561 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     4621 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4681 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     4741 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4801 aacaaaatat taacgtttac aggatctaat gaggggacga cgacagtatc ggcctcagga
     4861 agatcgcact ccagccagct ttccggcacc gcttctggtg ccggaaacca ggcaaagcgc
     4921 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     4981 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     5041 ttttcccagt cacgacgttg taaaacgacg gccagtgaat ccgtaatcat ggtcatagct
     5101 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5161 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5221 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5281 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     5341 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     5401 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttgacggc gggatataac
     5461 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     5521 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     5581 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     5641 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     5701 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     5761 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     5821 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     5881 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     5941 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     6001 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     6061 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     6121 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     6181 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     6241 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     6301 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     6361 tgcaccattc catggtgtcg aattgctgca ggtcgagggg gtcatggctg cgccccgaca
     6421 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     6481 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     6541 acgcgcgagg cagcaaggag atggcgccca acagtccccc ggccacgggc ctgccaccat
     6601 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     6661 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     6721 gcgtccggcg tagaggatcc ggagcttatc gactgcacgg tgcaccaatg cttctggcgt
     6781 caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt
     6841 gtcgctcaag gcgcactccc gttctggata atgttttttg cgccga
//
