LOCUS       sequence-221946-        5759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express periplasmic, outer membrane, or secreted proteins in E. coli
            - TamA signal and His6 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5759)
  AUTHORS   Casasanta MA, Slade DJ
  TITLE     A Vector Suite for the Overexpression and Purification of Tagged 
            Outer Membrane, Periplasmic, and Secreted Proteins in E. coli.
  JOURNAL   Methods Mol Biol. 2019;1960:123-138. doi: 
            10.1007/978-1-4939-9167-9_11.
  PUBMED    30798527
REFERENCE   2  (bases 1 to 5759)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods Mol
            Biol."; date: "2019"; volume: "1960:123-138. doi"; pages: " 
            10.1007/978-1-4939-9167-9_11"
COMMENT     Sequence Label: pDJSVT89
FEATURES             Location/Qualifiers
     source          1..5759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(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"
     RBS             453..475
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    490..514
                     /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(514..533)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(515..533)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(617..636)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        842..919
                     /gene="lacI"
                     /label=lacI promoter
     CDS             920..2002
                     /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(939..958)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3314..3505
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3499..3521)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3607..3746
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3662..3681
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3761..3778)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3932..4520)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4012..4031)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4691..5482,5483..5551))
                     /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     5314..5333
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5552..5656)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5724..5742
                     /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 tgctgtagac ggacgttcgc ggcgacggca gatccgctta agcagagtaa gcttacacag
      421 cataactgtc ggatatagcg tcccatggta tatctccttc ttaaagttaa acaaaattat
      481 ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc
      541 gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
      601 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
      661 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
      721 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc
      781 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc
      841 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
      901 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
      961 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     1021 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     1081 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     1141 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     1201 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     1261 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     1321 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     1381 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     1441 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     1501 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     1561 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     1621 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     1681 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
     1741 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     1801 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     1861 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     1921 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     1981 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     2041 caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     2101 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     2161 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     2221 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     2281 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     2341 ccgacgcgct gggctacgtc ttgctggcgt tcgcgacgcg aggctggatg gccttcccca
     2401 ttatgattct tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc atgctgtcca
     2461 ggcaggtaga tgacgaccat cagggacagc ttcaaggatc gctcgcggct cttaccagcc
     2521 taacttcgat cattggaccg ctgatcgtca cggcgattta tgccgcctcg gcgagcacat
     2581 ggaacgggtt ggcatggatt gtaggcgccg ccctatacct tgtctgcctc cccgcgttgc
     2641 gtcgcggtgc atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa
     2701 cggattcacc actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca
     2761 aaccaaccct tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg
     2821 catctcgggc agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     2881 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     2941 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     3001 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     3061 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     3121 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     3181 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     3241 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     3301 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     3361 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     3421 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
     3481 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     3541 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     3601 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     3661 gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata ccgcacagat
     3721 gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc
     3781 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3841 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3901 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3961 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4021 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4081 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4141 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4201 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4261 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4321 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     4381 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4441 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4501 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4561 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4621 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4681 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4741 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4801 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4861 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4921 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4981 gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta
     5041 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5101 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5161 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5221 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5281 gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt
     5341 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5401 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5461 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5521 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5581 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5641 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     5701 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaa
//
