LOCUS       sequence-257540-        6990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Allows Gateway cloning into the bacterial 6xHIS tagged protein 
            expression vector pET26.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6990)
  TITLE     Feher Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6990)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pETDest
FEATURES             Location/Qualifiers
     source          1..6990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             32..54
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    69..93
                     /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(93..112)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(94..112)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(196..215)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        421..498
                     /gene="lacI"
                     /label=lacI promoter
     CDS             499..1581
                     /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(518..537)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2390..2581
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2575..2597)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2683..2822
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2738..2757
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2837..2854)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3008..3596)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3088..3107)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3707..3726)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3718..4533
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             3718..4533
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3791..3810)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4625..5080)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4762..4783)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4974..4993
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5117..5164)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5160..5178
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5231..5248)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    5271..5395
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(5436..5741)
                     /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(5538..5557)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(6083..6742)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6657..6676
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(6796..6802,6803..6820,6821..6826))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(6851..6975)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(join(6985..6990,1..12))
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 gtgatggtga tggtagtacg acatatgtat atctccttct taaagttaaa caaaattatt
       61 tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg
      121 ggatcgagat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag
      181 gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact
      241 tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac
      301 tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca
      361 acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg
      421 gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
      481 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
      541 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
      601 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
      661 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
      721 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
      781 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
      841 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
      901 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
      961 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     1021 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     1081 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     1141 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     1201 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     1261 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     1321 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     1381 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     1441 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     1501 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     1561 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc
     1621 accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc
     1681 gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg
     1741 acaggtgccg gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac
     1801 gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt
     1861 cactggtccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc
     1921 cccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg
     1981 ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg
     2041 caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag
     2101 tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg
     2161 ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg
     2221 agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc
     2281 cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat
     2341 cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca
     2401 ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga
     2461 gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca
     2521 cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     2581 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     2641 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     2701 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
     2761 agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     2821 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2881 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2941 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3001 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3061 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3121 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3181 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3241 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3301 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3361 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3421 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     3481 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3541 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3601 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     3661 ttttggtcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg
     3721 agccatattc aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct
     3781 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     3841 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     3901 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     3961 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     4021 cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     4081 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     4141 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     4201 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     4261 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     4321 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     4381 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     4441 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     4501 cagtttcatt tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt
     4561 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     4621 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     4681 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     4741 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     4801 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     4861 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     4921 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     4981 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5041 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc aatccggata
     5101 tagttcctcc tttcagcaaa aaacccctca agacccgttt agaggcccca aggggttatg
     5161 ctagttattg ctcagcggtg gcagcagcca actcagcttc ctttcgggct ttgttagcag
     5221 ccggatctca gtggtggtgg tggtggtgct cgagtagcag cctcgaatca accactttgt
     5281 acaagaaagc tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt
     5341 tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca ctatggtcga
     5401 cctgcagact ggctgtgtat aagggagcct gacatttata ttccccagaa catcaggtta
     5461 atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc cccgataacg
     5521 gagaccggca cactggccat atcggtggtc atcatgcgcc agctttcatc cccgatatgc
     5581 accaccgggt aaagttcacg ggagacttta tctgacagca gacgtgcact ggccaggggg
     5641 atcaccatcc gtcgcccggg cgtgtcaata atatcactct gtacatccac aaacagacga
     5701 taacggctct ctcttttata ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc
     5761 gtcagcaaaa gagccgttca tttcaataaa ccgggcgacc tcagccatcc cttcctgatt
     5821 ttccgctttc cagcgttcgg cacgcagacg acgggcttca ttctgcatgg ttgtgcttac
     5881 cagaccggag atattgacat catatatgcc ttgagcaact gatagctgtc gctgtcaact
     5941 gtcactgtaa tacgctgctt catagcatac ctctttttga catacttcgg gtatacatat
     6001 cagtatatat tcttataccg caaaaatcag cgcgcaaata cgcatactgt tatctggctt
     6061 ttagtaagcc ggatccagat ctttacgccc cgccctgcca ctcatcgcag tactgttgta
     6121 attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg
     6181 ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg
     6241 cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat
     6301 tggctgagac gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac
     6361 cgtaacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt
     6421 cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa
     6481 cactatccca tatcaccagc tcaccgtctt tcattgccat acggaattcc ggatgagcat
     6541 tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta
     6601 cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa
     6661 ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat
     6721 atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc gacggatcct
     6781 aactcaaaat ccacacatta tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     6841 atgcggccgc catagtgact ggatatgttg tgttttacag tattatgtag tctgtttttt
     6901 atgcaaaatc taatttaata tattgatatt tatatcattt tacgtttctc gttcagcttt
     6961 tttgtacaaa cttgttgatt cgaggtgatg
//
