LOCUS       sequence-8812-ex        7934 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET19b converted Gateway Destination vector for expressing His 
            tagged fusion proteins in bacteria. See note in comments section 
            below regarding the mutated enterokinase site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7934)
  AUTHORS   Guo F, Chiang MY, Wang Y, Zhang YZ
  TITLE     An in vitro recombination method to convert restriction- and 
            ligation-independent expression vectors.
  JOURNAL   Biotechnol J. 2008 Mar . 3(3):370-7.
  PUBMED    18064608
REFERENCE   2  (bases 1 to 7934)
  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: "Biotechnol 
            J. 2008 Mar . 3(3):370-7."
COMMENT     Sequence Label: pEZY19
FEATURES             Location/Qualifiers
     source          1..7934
                     /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"
     protein_bind    279..403
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        581..683
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             684..1343
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(750..769)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1688..1993
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1872..1891
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2422..2546)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(2553..2567)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(2583..2612)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             2625..2647
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2662..2686
                     /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(2686..2705)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2687..2705)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2789..2808)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3014..3091
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3092..4174
                     /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(3111..3130)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5486..5677
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5671..5693)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5779..5921
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5834..5853
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5936..5953)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6107..6695)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6187..6206)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6866..7657,7658..7726))
                     /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     7489..7508
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7727..7831)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7899..7917
                     /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 gctcgaggac cactttgtac aagaaagctg
      301 aacgagaaac gtaaaatgat ataaatatca atatattaaa ttagattttg cataaaaaac
      361 agactacata atactgtaaa acacaacata tccagtcact atgaatcaac tacttagatg
      421 gtattagtga cctgtagtcg actaagttgg cagcatcacc cgacgcactt tgcgccgaat
      481 aaatacctgt gacggaagat cacttcgcag aataaataaa tcctggtgtc cctgttgata
      541 ccgggaagcc ctgggccaac ttttggcgaa aatgagacgt tgatcggcac gtaagaggtt
      601 ccaactttca ccataatgaa ataagatcac taccgggcgt attttttgag ttatcgagat
      661 tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata
      721 tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct
      781 ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc
      841 acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat
      901 tccgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca
      961 ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt
     1021 tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct
     1081 atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt
     1141 tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca
     1201 tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc
     1261 atgccgtctg tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg
     1321 atgagtggca gggcggggcg taaacgccgc gtggatccgg cttactaaaa gccagataac
     1381 agtatgcgta tttgcgcgct gatttttgcg gtataagaat atatactgat atgtataccc
     1441 gaagtatgtc aaaaagaggt atgctatgaa gcagcgtatt acagtgacag ttgacagcga
     1501 cagctatcag ttgctcaagg catatatgat gtcaatatct ccggtctggt aagcacaacc
     1561 atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa tcaggaaggg
     1621 atggctgagg tcgcccggtt tattgaaatg aacggctctt ttgctgacga gaacaggggc
     1681 tggtgaaatg cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt
     1741 ggatgtacag agtgatatta ttgacacgcc cgggcgacgg atggtgatcc ccctggccag
     1801 tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga
     1861 tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg ttatcgggga
     1921 agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt
     1981 ctggggaata taaatgtcag gctcccttat acacagccag tctgcaggtc gatacagtag
     2041 aaattacaga aactttatca cgtttagtaa gtatagaggc tgaaaatcca gatgaagccg
     2101 aacgacttgt aagagaaaag tataagagtt gtgaaattgt tcttgatgca gatgattttc
     2161 aggactatga cactagcgta tatgaatagg tagatgtttt tattttgtca cacaaaaaag
     2221 aggctcgcac ctctttttct tatttctttt tatgatttaa tacggcattg aggacaatag
     2281 cgagtaggct ggatacgacg attccgtttg agaagaacat ttggaaggct gtcggtcgac
     2341 taagttggca gcatcacccg aagaacattt ggaaggctgt cggtcgacta caggtcacta
     2401 ataccatcta agtagttgat tcatagtgac tggatatgtt gtgttttaca gtattatgta
     2461 gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct
     2521 cgttcagctt ttttgtacaa acttgtatta tgcttgtcgt cgtcgtcgat atggccgctg
     2581 ctgtgatgat gatgatgatg atgatgatga tggcccatgg tatatctcct tcttaaagtt
     2641 aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg
     2701 tattaatttc gcgggatcga gatctcgatc ctctacgccg gacgcatcgt ggccggcatc
     2761 accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat
     2821 cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc
     2881 gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg
     2941 ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag
     3001 cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg
     3061 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     3121 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     3181 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
     3241 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
     3301 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
     3361 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
     3421 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
     3481 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
     3541 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
     3601 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
     3661 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
     3721 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
     3781 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
     3841 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt
     3901 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
     3961 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
     4021 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     4081 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     4141 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc
     4201 tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct
     4261 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca
     4321 tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc
     4381 gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg
     4441 ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg
     4501 ccggcatggc ggccgacgcg ctgggctacg tcttgctggc gttcgcgacg cgaggctgga
     4561 tggccttccc cattatgatt cttctcgctt ccggcggcat cgggatgccc gcgttgcagg
     4621 ccatgctgtc caggcaggta gatgacgacc atcagggaca gcttcaagga tcgctcgcgg
     4681 ctcttaccag cctaacttcg atcactggac cgctgatcgt cacggcgatt tatgccgcct
     4741 cggcgagcac atggaacggg ttggcatgga ttgtaggcgc cgccctatac cttgtctgcc
     4801 tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg gaagccggcg
     4861 gcacctcgct aacggattca ccactccaag aattggagcc aatcaattct tgcggagaac
     4921 tgtgaatgcg caaaccaacc cttggcagaa catatccatc gcgtccgcca tctccagcag
     4981 ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca tgatcgtgct
     5041 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc
     5101 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac
     5161 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc
     5221 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc
     5281 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg
     5341 catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc
     5401 agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag
     5461 aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc
     5521 ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga
     5581 tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg
     5641 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     5701 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     5761 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     5821 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat atgcggtgtg
     5881 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     5941 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     6001 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     6061 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     6121 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     6181 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     6241 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6301 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6361 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6421 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6481 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6541 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6601 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6661 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6721 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6781 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6841 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6901 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6961 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     7021 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     7081 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     7141 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac
     7201 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     7261 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     7321 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7381 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7441 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc
     7501 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7561 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7621 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7681 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7741 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7801 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     7861 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     7921 ttcgtcttca agaa
//
