LOCUS       sequence-326570-        7885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector with N-terminal Trx-tag, 6X His-tag, 
            thrombin site, S-tag, and TEV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7885)
  TITLE     Arrowsmith Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7885)
  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            "Unpublished"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-LIC
FEATURES             Location/Qualifiers
     source          1..7885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        73..150
                     /gene="lacI"
                     /label=lacI promoter
     CDS             151..1233
                     /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(170..189)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2042..2233
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2227..2249)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2335..2474
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2390..2409
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2489..2506)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2660..3248)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2740..2759)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3419..4210,4211..4279))
                     /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     4042..4061
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4280..4384)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4410..4865)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4547..4568)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4759..4778
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4902..4949)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4945..4963
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5016..5033)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(5135..6469,6470..6556))
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        complement(6557..7002)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     protein_bind    7017..7050
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(7078..7098)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             complement(7114..7158)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(7165..7182)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(7192..7209)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(7231..7557)
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     RBS             7565..7587
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    7602..7626
                     /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(7626..7645)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7627..7645)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7733..7752)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
       61 gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
      121 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
      181 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
      241 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
      301 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
      361 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
      421 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
      481 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
      541 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
      601 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
      661 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
      721 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
      781 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
      841 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
      901 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga catctcggta
      961 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     1021 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     1081 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     1141 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     1201 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     1261 cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc
     1321 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     1381 gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg
     1441 ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc
     1501 tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc
     1561 cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg
     1621 ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag
     1681 cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg
     1741 tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg
     1801 catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg
     1861 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc
     1921 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct
     1981 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc
     2041 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt
     2101 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc
     2161 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
     2221 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     2281 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     2341 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     2401 cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa
     2461 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2521 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2581 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2641 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2701 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2761 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2821 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2881 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2941 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3001 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3061 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     3121 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3181 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3241 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3301 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3361 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3421 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3481 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3541 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3601 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3661 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3721 ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3781 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3841 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3901 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3961 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4021 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4081 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4141 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4201 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4261 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4321 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4381 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
     4441 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     4501 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     4561 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     4621 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     4681 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     4741 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     4801 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     4861 cgcgtcccat tcgccaatcc ggatatagtt cctcctttca gcaaaaaacc cctcaagacc
     4921 cgtttagagg ccccaagggg ttatgctagt tattgctcag cggtggcagc agccaactca
     4981 gcttcctttc gggctttgtt agcagccgga tctcagtggt ggtggtggtg gtgctcgagt
     5041 gcggccgcaa gcttcgtcat catgtgcatg atctcctcga aaagtgccac ctgacgtcaa
     5101 tgccaatagg atatcggcat tttcttttgc gtttttattt gttaactgtt aattgtcctt
     5161 gttcaaggat gctgtctttg acaacagatg ttttcttgcc tttgatgttc agcaggaagc
     5221 taggcgcaaa cgttgattgt ttgtctgcgt agaatcctct gtttgtcata tagcttgtaa
     5281 tcacgacatt gtttcctttc gcttgaggta cagcgaagtg tgagtaagta aaggttacat
     5341 cgttaggatc aagatccatt tttaacacaa ggccagtttt gttcagcggc ttgtatgggc
     5401 cagttaaaga attagaaaca taaccaagca tgtaaatatc gttagacgta atgccgtcaa
     5461 tcgtcatttt tgatccgcgg gagtcagtga acaggtacca tttgccgttc attttaaaga
     5521 cgttcgcgcg ttcaatttca tctgttactg tgttagatgc aatcagcggt ttcatcactt
     5581 ttttcagtgt gtaatcatcg tttagctcaa tcataccgag agcgccgttt gctaactcag
     5641 ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc ttgacggaag aatgatgtgc
     5701 ttttgccata gtatgctttg ttaaataaag attcttcgcc ttggtagcca tcttcagttc
     5761 cagtgtttgc ttcaaatact aagtatttgt ggcctttatc ttctacgtag tgaggatctc
     5821 tcagcgtatg gttgtcgcct gagctgtagt tgccttcatc gatgaactgc tgtacatttt
     5881 gatacgtttt tccgtcaccg tcaaagattg atttataatc ctctacaccg ttgatgttca
     5941 aagagctgtc tgatgctgat acgttaactt gtgcagttgt cagtgtttgt ttgccgtaat
     6001 gtttaccgga gaaatcagtg tagaataaac ggatttttcc gtcagatgta aatgtggctg
     6061 aacctgacca ttcttgtgtt tggtctttta ggatagaatc atttgcatcg aatttgtcgc
     6121 tgtctttaaa gacgcggcca gcgtttttcc agctgtcaat agaagtttcg ccgacttttt
     6181 gatagaacat gtaaatcgat gtgtcatccg catttttagg atctccggct aatgcaaaga
     6241 cgatgtggta gccgtgatag tttgcgacag tgccgtcagt gttttgtaat ggccagctgt
     6301 cccaaacgtc caggcctttt gcagaagaga tatttttaat tgtggacgaa tcgaactcag
     6361 gaactttata tttttcattt ttttgctgtt cagggatttg cagcatatca tggcgtgtaa
     6421 tatgggaaat gccgtatgtt tccttatatg gcttttggtt cgtttctttc gcaaacgctt
     6481 gagttgcgcc tcctgccagc agtgcggtag taaaggttaa tactgttgct tgttttgcaa
     6541 actttttgat gttcatcgtt catgtctcct tttttatgta ctgtgttagc ggtctgcttc
     6601 ttccagccct cctgtttgaa gatggcaagt tagttacgca caataaaaaa agacctaaaa
     6661 tatgtaaggg gtgacgccaa agtatacact ttgcccttta cacattttag gtcttgcctg
     6721 ctttatcagt aacaaacccg cgcgatttac ttttcgacct cattctatta gactctcgtt
     6781 tggattgcaa ctggtctatt ttcctctttt gtttgataga aaatcataaa aggatttgca
     6841 gactacgggc ctaaagaact aaaaaatcta tctgtttctt ttcattctct gtatttttta
     6901 tagtttctgt tgcatgggca taaagttgcc tttttaatca caattcagaa aatatcataa
     6961 tatctcattt cactaaataa tagtgaacgg caggtatatg tggacgtcgc ggccgcataa
     7021 cttcgtataa tgtatgctat acgaagttat ggatctttca ggaggagaac tcatatggct
     7081 ctggaaatac aagttttcgg tacccagatc tgggctgtcc atgtgctggc gttcgaattt
     7141 agcagcagcg gtttctttca taccagaacc gcgtggcacc agaccagaag aatgatgatg
     7201 atgatggtgc atatggccag aaccagaacc ggccaggtta gcgtcgagga actctttcaa
     7261 ctgaccttta gacagtgcac ccactttggt tgccgccact tcaccgtttt tgaacagcag
     7321 cagagtcggg ataccacgga tgccatattt cggcgcagtg ccagggtttt gatcgatgtt
     7381 cagttttgca acggtcagtt tgccctgata ttcgtcagcg atttcatcca gaatcggggc
     7441 gatcattttg cacggaccgc accactctgc ccagaaatcg acgaggatcg ccccgtccgc
     7501 tttgagtaca tccgtgtcaa aactgtcgtc agtcaggtga ataattttat cgctcatatg
     7561 tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac
     7621 aattccccta tagtgagtcg tattaatttc gcgggatcga gatcgatctc gatcctctac
     7681 gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc
     7741 gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc
     7801 ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat
     7861 gcaccattcc ttgcggcggc ggtgc
//
