LOCUS       sequence-8813-ex        7522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pKW32 converted Gateway Destination vector for expressing hemoglobin
            tagged fusion proteins in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7522)
  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 7522)
  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: pEZYHb
FEATURES             Location/Qualifiers
     source          1..7522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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)."
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             616..633
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     protein_bind    643..767
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1196..1501)
                     /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     complement(1298..1317)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1846..2505)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(2506..2608)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2786..2910)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      2977..3071
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             3115..3774
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     terminator      3839..3925
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4018..5100)
                     /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     5062..5081
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5101..5178)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(5262..5284)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5370..5512
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5425..5444
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5527..5544)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5698..6286)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5778..5797)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6457..7248,7249..7317))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7080..7099
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7318..7422)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7490..7508
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagta ttcattaaag aggagaaatt aactatgaga
      121 ggatcgcatc accatcacca tcacggatcc atgttagacc agcaaaccat taacatcatc
      181 aaagccactg ttcctgtatt gaaggagcat ggcgttacca ttaccacgac tttttataaa
      241 aacttgtttg ccaaacaccc tgaagtacgt cctttgtttg atatgggtcg ccaagaatct
      301 ttggagcagc ctaaggcttt ggcgatgacg gtattggcgg cagcgcaaaa cattgaaaat
      361 ttgccagcta ttttgcctgc ggtcaaaaaa attgcagtca aacattgtca agcaggcgtg
      421 gcagcagcgc attatccgat tgtcggtcaa gaattgttgg gtgcgattaa agaagtattg
      481 ggcgatgccg caaccgatga cattttggac gcgtggggca aggcttatgg cgtgattgca
      541 gatgtgttta ttcaagtgga agcagatttg tacgctcaag cggttgaacc aggaatatca
      601 ggcggaggcg gaggcttggt ccctcgaggg agtgaacata atacaagttt gtacaaaaaa
      661 gctgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa
      721 aaacagacta cataatactg taaaacacaa catatccagt cactatgaat caactactta
      781 gatggtatta gtgacctgta gtcgaccgac agccttccaa atgttcttcg ggtgatgctg
      841 ccaacttagt cgaccgacag ccttccaaat gttcttctca aacggaatcg tcgtatccag
      901 cctactcgct attgtcctca atgccgtatt aaatcataaa aagaaataag aaaaagaggt
      961 gcgagcctct tttttgtgtg acaaaataaa aacatctacc tattcatata cgctagtgtc
     1021 atagtcctga aaatcatctg catcaagaac aatttcacaa ctcttatact tttctcttac
     1081 aagtcgttcg gcttcatctg gattttcagc ctctatactt actaaacgtg ataaagtttc
     1141 tgtaatttct actgtatcga cctgcagact ggctgtgtat aagggagcct gacatttata
     1201 ttccccagaa catcaggtta atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag
     1261 ccacttcttc cccgataacg gagaccggca cactggccat atcggtggtc atcatgcgcc
     1321 agctttcatc cccgatatgc accaccgggt aaagttcacg ggagacttta tctgacagca
     1381 gacgtgcact ggccaggggg atcaccatcc gtcgcccggg cgtgtcaata atatcactct
     1441 gtacatccac aaacagacga taacggctct ctcttttata ggtgtaaacc ttaaactgca
     1501 tttcaccagc ccctgttctc gtcagcaaaa gagccgttca tttcaataaa ccgggcgacc
     1561 tcagccatcc cttcctgatt ttccgctttc cagcgttcgg cacgcagacg acgggcttca
     1621 ttctgcatgg ttgtgcttac cagaccggag atattgacat catatatgcc ttgagcaact
     1681 gatagctgtc gctgtcaact gtcactgtaa tacgctgctt catagcatac ctctttttga
     1741 catacttcgg gtatacatat cagtatatat tcttataccg caaaaatcag cgcgcaaata
     1801 cgcatactgt tatctggctt ttagtaagcc ggatccacgc ggcgtttacg ccccgccctg
     1861 ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca
     1921 gacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata
     1981 tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa
     2041 actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt
     2101 agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa
     2161 ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg
     2221 gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc
     2281 catacggaat tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata
     2341 aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt
     2401 ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca
     2461 ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc
     2521 tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg
     2581 aaagttggaa cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg
     2641 cttcccggta tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac
     2701 aggtatttat tcggcgcaaa gtgcgtcggg tgatgctgcc aacttagtcg actacaggtc
     2761 actaatacca tctaagtagt tgattcatag tgactggata tgttgtgttt tacagtatta
     2821 tgtagtctgt tttttatgca aaatctaatt taatatattg atatttatat cattttacgt
     2881 ttctcgttca gctttcttgt acaaagtggt cctcgacggc ggccgcgaat tcctcgagcc
     2941 cgggtgactg cagccaagct taattagctg agcttggact cctgttgata gatccagtaa
     3001 tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg gcgtttttta
     3061 ttggtgagaa tccaagctag cttggcgaga ttttcaggag ctaaggaagc taaaatggag
     3121 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
     3181 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
     3241 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
     3301 cttgcccgcc tgatgaatgc tcatccggaa tttcgtatgg caatgaaaga cggtgagctg
     3361 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
     3421 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     3481 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     3541 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     3601 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     3661 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
     3721 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt
     3781 taaggcagtt attggtgccc ttaaacgcct ggggtaatga ctctctagct tgaggcatca
     3841 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     3901 gaacgctctc ctgagtagga caaatccgcc ctctagatta cgtgcagtcg atgataagct
     3961 gtcaaacatg agaattgtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     4021 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4081 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     4141 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     4201 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     4261 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     4321 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     4381 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     4441 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     4501 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     4561 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     4621 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     4681 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     4741 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     4801 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     4861 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     4921 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     4981 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     5041 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     5101 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     5161 gcaccattcg atggtgtcgg aatttcgggc agcgttgggt cctggccacg ggtgcgcatg
     5221 atctagagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     5281 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     5341 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     5401 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     5461 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
     5521 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5581 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5641 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5701 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5761 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5821 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5881 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5941 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6001 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6061 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6121 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6181 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6241 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6301 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6361 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6421 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6481 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6541 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6601 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6661 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6721 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6781 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6841 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6901 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6961 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7021 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     7081 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7141 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7201 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7261 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7321 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7381 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7441 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     7501 cacgaggccc tttcgtcttc ac
//
