LOCUS       40924_33777        5588 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pOPING-ET, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5588)
  AUTHORS   Nettleship JE, Ren J, Rahman N, Berrow NS, Hatherley D, Barclay AN, 
            Owens RJ.
  TITLE     A pipeline for the production of antibody fragments for structural 
            studies using transient expression in HEK 293T cells
  JOURNAL   Protein Expr. Purif. 62 (1), 83-89 (2008)
  PUBMED    18662785
REFERENCE   2  (bases 1 to 5588)
  AUTHORS   Berrow NS, Owens RJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (20-MAY-2008) Oxford Protein Production Facility, Oxford 
            University, Roosevelt Drive, Oxford, Oxfordshire OX3 7BN, UK
REFERENCE   3  (bases 1 to 5588)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5588)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Protein 
            Expr. Purif."; date: "2008"; volume: "62"; issue: "1"; pages: 
            "83-89"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (20-MAY-2008) Oxford Protein Production Facility, Oxford University,
            Roosevelt Drive, Oxford, Oxfordshire OX3 7BN, UK"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5588
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     enhancer        1140..1443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1449..1726
                     /label=chicken beta-actin promoter
     promoter        2150..2168
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2169..2193
                     /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        2209..2318
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     misc_feature    2333..2416
                     /label=mu-phosphatase secretion leader peptide
                     /note="mu-phosphatase secretion leader peptide"
     misc_feature    2403..2417
                     /label=5' infusion site after KpnI digest
                     /note="5' infusion site after KpnI digest"
     protein_bind    2463..2484
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2499..2529
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2537..2553
                     /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)."
     CDS             2573..2746
                     /codon_start=1
                     /label=lacZ-alpha
                     /note="LacZ-alpha fragment of beta-galactosidase"
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     CDS             2776..2793
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     polyA_signal    2948..3003
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     terminator      3091..3138
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3151..3856
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     protein_bind    complement(3865..3898)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(4066..4653)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4669..5526)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggccg
     1021 taatgagacg cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctct
     1081 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     1141 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     1201 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     1261 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     1321 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     1381 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     1441 atgcatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     1501 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     1561 gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg
     1621 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
     1681 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1741 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1801 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctccttcgg gctgtaatta
     1861 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
     1921 ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc
     1981 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag
     2041 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
     2101 ttattgtgct gtctcatcat tttggcaaag aattggatcg gaccgaaatt aatacgactc
     2161 actatagggg aattgtgagc ggataacaat tccccggagt taatccggga cctttaattc
     2221 aacccaacac aatatattat agttaaataa gaattattat caaatcattt gtatattaat
     2281 taaaatacta tactgtaaat tacattttat ttacaatcaa aggagatata ccatggggat
     2341 ccttcccagc cctgggatgc ctgcgctgct ctccctcgtg agccttctct ccgtgctgct
     2401 gatgggttgc gtagctggta ccgtcgaccc gactggaaag cgggcagtga gcgcaacgca
     2461 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     2521 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     2581 gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     2641 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     2701 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gccaagcttt
     2761 ctagaccagt ttaaacatca ccatcaccat cactaagtga ttaacctcag gtgcaggctg
     2821 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc
     2881 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     2941 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     3001 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     3061 tagagtttgg caacatatgc ccatatgtaa ctagcataac cccttggggc ctctaaacgg
     3121 gtcttgaggg gttttttgct gaaagcatgc ggaggaaatt ctccttgaag tttccctggt
     3181 gttcaaagta aaggagtttg caccagacgc acctctgttc actggtccgg cgtattaaaa
     3241 cacgatacat tgttattagt acatttatta agcgctagat tctgtgcgtt gttgatttac
     3301 agacaattgt tgtacgtatt ttaataattc attaaattta taatctttag ggtggtatgt
     3361 tagagcgaaa atcaaatgat tttcagcgtc tttatatctg aatttaaata ttaaatcctc
     3421 aatagatttg taaaataggt ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat
     3481 ggctggacta tctaatggat tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag
     3541 caatctagct ttgtcgatat tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt
     3601 caaaatatta tgcgcttttg tatttctttc atcactgtcg ttagtgtaca attgactcga
     3661 cgtaaacacg ttaaatagag cttggacata tttaacatcg ggcgtgttag ctttattagg
     3721 ccgattatcg tcgtcgtccc aaccctcgtc gttagaagtt gcttccgaag acgattttgc
     3781 catagccaca cgacgcctat taattgtgtc ggctaacacg tccgcgatca aatttgtagt
     3841 tgagcttttt ggaattgcga tcgcataact tcgtatagca tacattatac gaagttataa
     3901 gctcggaacg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3961 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4021 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4081 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4141 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4201 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa
     4261 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4321 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4381 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4441 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4501 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4561 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4621 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgttacc aatgcttaat
     4681 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4741 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4801 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4861 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4921 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4981 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5041 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5101 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5161 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5221 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5281 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5341 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5401 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5461 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5521 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgtc
     5581 cgcgcgtt
//
