LOCUS       sequence-162124-        7611 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector derived from pET32 with Gateway cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7611)
  TITLE     Olszewski lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7611)
  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            "Unpublished"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: pET32a-DEST
FEATURES             Location/Qualifiers
     source          1..7611
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             50..67
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(120..138)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      134..181
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      218..673
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(305..324)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     515..536
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        700..804
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(805..873,874..1665)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1023..1042)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1836..2424
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2325..2344
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2578..2595
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2610..2752
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2678..2697)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2838..2860
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2854..3045)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3854..4936)
                     /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     4898..4917
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4937..5014)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5220..5239
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5327..5346
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5327..5345
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5346..5370
                     /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             5385..5407
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5415..5741
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             5763..5780
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5790..5807
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5814..5858
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             5874..5888
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     protein_bind    5904..6028
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(6053..6058,6059..6076,6077..6083)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             6137..6796
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(6203..6222)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             7138..7443
                     /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     7322..7341
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(7484..7608)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 atcggatccg aattcgagct ccgtcgacaa gcttgcggcc gcactcgagc accaccacca
       61 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
      121 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
      181 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
      241 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
      301 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
      361 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
      421 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
      481 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
      541 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
      601 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
      661 acgtttacaa tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
      721 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
      781 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
      841 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
      901 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
      961 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     1021 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     1081 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     1141 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     1201 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     1261 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     1321 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     1381 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     1441 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     1501 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     1561 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     1621 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     1681 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     1741 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     1801 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     1861 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     1921 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1981 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2041 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2101 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2161 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2221 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2281 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2341 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2401 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2461 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     2521 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     2581 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     2641 tgcggtattt cacaccgcat atatggtgca ctctcagtac aatctgctct gatgccgcat
     2701 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     2761 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     2821 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     2881 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     2941 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     3001 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     3061 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     3121 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     3181 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     3241 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     3301 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     3361 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     3421 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     3481 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     3541 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     3601 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     3661 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     3721 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     3781 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     3841 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3901 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     3961 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     4021 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     4081 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg
     4141 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     4201 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     4261 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     4321 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     4381 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     4441 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     4501 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     4561 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     4621 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     4681 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     4741 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     4801 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     4861 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     4921 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     4981 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     5041 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     5101 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     5161 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     5221 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     5281 atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg cgaaattaat acgactcact
     5341 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
     5401 aaggagatat acatatgagc gataaaatta ttcacctgac tgacgacagt tttgacacgg
     5461 atgtactcaa agcggacggg gcgatcctcg tcgatttctg ggcagagtgg tgcggtccgt
     5521 gcaaaatgat cgccccgatt ctggatgaaa tcgctgacga atatcagggc aaactgaccg
     5581 ttgcaaaact gaacatcgat caaaaccctg gcactgcgcc gaaatatggc atccgtggta
     5641 tcccgactct gctgctgttc aaaaacggtg aagtggcggc aaccaaagtg ggtgcactgt
     5701 ctaaaggtca gttgaaagag ttcctcgacg ctaacctggc cggttctggt tctggccata
     5761 tgcaccatca tcatcatcat tcttctggtc tggtgccacg cggttctggt atgaaagaaa
     5821 ccgctgctgc taaattcgaa cgccagcaca tggacagccc agatctgggt accgacgacg
     5881 acgacaaggc catggctgat atcacaagtt tgtacaaaaa agctgaacga gaaacgtaaa
     5941 atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact
     6001 gtaaaacaca acatatccag tcactatggc ggccgcatta ggcaccccag gctttacact
     6061 ttatgcttcc ggctcgtata atgtgtggat tttgagttag gatccgtcga gattttcagg
     6121 agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca
     6181 atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca
     6241 gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt
     6301 ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg aattccgtat
     6361 ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt
     6421 ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca
     6481 gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc
     6541 taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag
     6601 ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa
     6661 atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt
     6721 ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg
     6781 gcagggcggg gcgtaaacgc gtggatccgg cttactaaaa gccagataac agtatgcgta
     6841 tttgcgcgct gatttttgcg gtataagaat atatactgat atgtataccc gaagtatgtc
     6901 aaaaagaggt atgctatgaa gcagcgtatt acagtgacag ttgacagcga cagctatcag
     6961 ttgctcaagg catatatgat gtcaatatct ccggtctggt aagcacaacc atgcagaatg
     7021 aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa tcaggaaggg atggctgagg
     7081 tcgcccggtt tattgaaatg aacggctctt ttgctgacga gaacaggggc tggtgaaatg
     7141 cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag
     7201 agtgatatta ttgacacgcc cgggcgacgg atggtgatcc ccctggccag tgcacgtctg
     7261 ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg
     7321 cgcatgatga ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct
     7381 gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata
     7441 taaatgtcag gctcccttat acacagccag tctgcaggtc gaccatagtg actggatatg
     7501 ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta atatattgat
     7561 atttatatca ttttacgttt ctcgttcagc tttcttgtac aaagtggtga t
//
