LOCUS       Exported                5762 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Express periplasmic, outer membrane, or secreted proteins in E. coli
            - OmpA signal and 6His tag.
ACCESSION   .
VERSION     .
KEYWORDS    pDJSVT86
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5762)
  AUTHORS   Casasanta MA, Yoo CC, Smith HB, Duncan AJ, Cochrane K, Varano AC, 
            Allen-Vercoe E, Slade DJ
  TITLE     A chemical and biological toolbox for Type Vd secretion: 
            Characterization of the phospholipase A1 autotransporter FplA from 
            Fusobacterium nucleatum.
  JOURNAL   J Biol Chem. 2017 Dec 8;292(49):20240-20254. doi: 
            10.1074/jbc.M117.819144. Epub 2017 Oct 11.
  PUBMED    29021252
REFERENCE   2  (bases 1 to 5762)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5762
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        10..38
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      213..260
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     256..274
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(345..362)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     sig_peptide     complement(381..443)
                     /product="signal peptide from the E. coli outer membrane 
                     protein OmpA"
                     /label=OmpA signal peptide
     RBS             456..478
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    493..517
                     /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)."
     primer_bind     complement(517..536)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(518..536)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(620..639)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        845..922
                     /gene="lacI"
                     /label=lacI promoter
     CDS             923..2005
                     /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"
     primer_bind     complement(942..961)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3317..3508
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3502..3524)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3610..3749
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3665..3684
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3764..3781)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3935..4523)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4015..4034)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4694..5554)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5317..5336
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5555..5659)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5727..5745
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
      241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
      301 cgggctttgt tagcagccgg atcctcgagg gtacccgcgg ccgcgtggtg gtggtggtgg
      361 tgggtgttat ctttcggagc ggcctgcgct acggtagcga aaccagccag tgccactgca
      421 atcgcgatag ctgtcttttt cattcccatg gtatatctcc ttcttaaagt taaacaaaat
      481 tatttctaga ggggaattgt tatccgctca caattcccct atagtgagtc gtattaattt
      541 cgcgggatcg agatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc
      601 acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc
      661 cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
      721 ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc
      781 ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat
      841 cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
      901 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
      961 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     1021 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     1081 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     1141 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     1201 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     1261 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     1321 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     1381 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     1441 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     1501 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     1561 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     1621 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     1681 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     1741 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     1801 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     1861 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     1921 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     1981 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     2041 aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     2101 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     2161 taggacaggt gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg
     2221 cgacgatgat cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct
     2281 tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg
     2341 cggccgacgc gctgggctac gtcttgctgg cgttcgcgac gcgaggctgg atggccttcc
     2401 ccattatgat tcttctcgct tccggcggca tcgggatgcc cgcgttgcag gccatgctgt
     2461 ccaggcaggt agatgacgac catcagggac agcttcaagg atcgctcgcg gctcttacca
     2521 gcctaacttc gatcattgga ccgctgatcg tcacggcgat ttatgccgcc tcggcgagca
     2581 catggaacgg gttggcatgg attgtaggcg ccgccctata ccttgtctgc ctccccgcgt
     2641 tgcgtcgcgg tgcatggagc cgggccacct cgacctgaat ggaagccggc ggcacctcgc
     2701 taacggattc accactccaa gaattggagc caatcaattc ttgcggagaa ctgtgaatgc
     2761 gcaaaccaac ccttggcaga acatatccat cgcgtccgcc atctccagca gccgcacgcg
     2821 gcgcatctcg ggcagcgttg ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt
     2881 gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg
     2941 cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat
     3001 ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat
     3061 tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt
     3121 attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac
     3181 cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg
     3241 tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc
     3301 ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat
     3361 cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc
     3421 agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg
     3481 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     3541 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     3601 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac
     3661 tatgcggcat cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca
     3721 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     3781 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3841 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3901 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3961 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4021 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4081 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4141 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4201 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4261 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4321 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     4381 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4441 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4501 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4561 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4621 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4681 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4741 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4801 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4861 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4921 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4981 atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg
     5041 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5101 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5161 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5221 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5281 ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa
     5341 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5401 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5461 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5521 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5581 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5641 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     5701 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcaag
     5761 aa
//
