LOCUS       sequence-102191-        7170 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector with N-terminal Hexa-HIS and TEV cleavage.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7170)
  TITLE     Arrowsmith Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7170)
  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            "Unpublished"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: pET21a-LIC
FEATURES             Location/Qualifiers
     source          1..7170
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             join(260..328,329..1120)
                     /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(478..497)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1291..1879
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1780..1799
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2033..2050
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2065..2207
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2133..2152)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2293..2315
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2309..2500)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3309..4391)
                     /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     4353..4372
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4392..4469)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4675..4694
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4782..4801
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4782..4800
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4801..4825
                     /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             4840..4862
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4873..4890
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4915..4935
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     protein_bind    complement(4965..4998)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5013..5458
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(5459..5545,5546..6880)
                     /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
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     CDS             7014..7031
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7084..7102)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7098..7145
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
      301 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
      361 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
      421 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
      481 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
      541 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
      601 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
      661 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
      721 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
      781 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
      841 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
      901 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
      961 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     1021 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     1081 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     1141 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     1201 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     1261 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     1321 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     1381 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     1441 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     1501 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     1561 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     1621 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     1681 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     1741 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     1801 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     1861 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     1921 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     1981 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     2041 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     2101 tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     2161 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     2221 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     2281 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     2341 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     2401 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     2461 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     2521 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     2581 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     2641 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     2701 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     2761 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     2821 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     2881 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     2941 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     3001 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     3061 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     3121 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     3181 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     3241 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     3301 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3361 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     3421 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     3481 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     3541 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     3601 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     3661 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     3721 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     3781 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     3841 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     3901 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     3961 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     4021 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     4081 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     4141 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     4201 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     4261 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     4321 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     4381 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     4441 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     4501 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     4561 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     4621 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     4681 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     4741 tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac tcactatagg
     4801 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     4861 gatatacata tgcaccatca tcatcatcat tcttctggtg tagatctggg taccgagaac
     4921 ctgtacttcc aatccataag ctagcttctc ctcctgaaag atccataact tcgtatagca
     4981 tacattatac gaagttatgc ggccgcgacg tccacatata cctgccgttc actattattt
     5041 agtgaaatga gatattatga tattttctga attgtgatta aaaaggcaac tttatgccca
     5101 tgcaacagaa actataaaaa atacagagaa tgaaaagaaa cagatagatt ttttagttct
     5161 ttaggcccgt agtctgcaaa tccttttatg attttctatc aaacaaaaga ggaaaataga
     5221 ccagttgcaa tccaaacgag agtctaatag aatgaggtcg aaaagtaaat cgcgcgggtt
     5281 tgttactgat aaagcaggca agacctaaaa tgtgtaaagg gcaaagtgta tactttggcg
     5341 tcacccctta catattttag gtcttttttt attgtgcgta actaacttgc catcttcaaa
     5401 caggagggct ggaagaagca gaccgctaac acagtacata aaaaaggaga catgaacgat
     5461 gaacatcaaa aagtttgcaa aacaagcaac agtattaacc tttactaccg cactgctggc
     5521 aggaggcgca actcaagcgt ttgcgaaaga aacgaaccaa aagccatata aggaaacata
     5581 cggcatttcc catattacac gccatgatat gctgcaaatc cctgaacagc aaaaaaatga
     5641 aaaatatcaa gttcctgagt tcgattcgtc cacaattaaa aatatctctt ctgcaaaagg
     5701 cctggacgtt tgggacagct ggccattaca aaacgctgac ggcactgtcg caaactatca
     5761 cggctaccac atcgtctttg cattagccgg agatcctaaa aatgcggatg acacatcgat
     5821 ttacatgttc tatcaaaaag tcggcgaaac ttctattgac agctggaaaa acgctggccg
     5881 cgtctttaaa gacagcgaca aattcgatgc aaatgattct atcctaaaag accaaacaca
     5941 agaatggtca ggttcagcca catttacatc tgacggaaaa atccgtttat tctacactga
     6001 tttctccggt aaacattacg gcaaacaaac actgacaact gcacaagtta acgtatcagc
     6061 atcagacagc tctttgaaca tcaacggtgt agaggattat aaatcaatct ttgacggtga
     6121 cggaaaaacg tatcaaaatg tacagcagtt catcgatgaa ggcaactaca gctcaggcga
     6181 caaccatacg ctgagagatc ctcactacgt agaagataaa ggccacaaat acttagtatt
     6241 tgaagcaaac actggaactg aagatggcta ccaaggcgaa gaatctttat ttaacaaagc
     6301 atactatggc aaaagcacat cattcttccg tcaagaaagt caaaaacttc tgcaaagcga
     6361 taaaaaacgc acggctgagt tagcaaacgg cgctctcggt atgattgagc taaacgatga
     6421 ttacacactg aaaaaagtga tgaaaccgct gattgcatct aacacagtaa cagatgaaat
     6481 tgaacgcgcg aacgtcttta aaatgaacgg caaatggtac ctgttcactg actcccgcgg
     6541 atcaaaaatg acgattgacg gcattacgtc taacgatatt tacatgcttg gttatgtttc
     6601 taattcttta actggcccat acaagccgct gaacaaaact ggccttgtgt taaaaatgga
     6661 tcttgatcct aacgatgtaa cctttactta ctcacacttc gctgtacctc aagcgaaagg
     6721 aaacaatgtc gtgattacaa gctatatgac aaacagagga ttctacgcag acaaacaatc
     6781 aacgtttgcg cctagcttcc tgctgaacat caaaggcaag aaaacatctg ttgtcaaaga
     6841 cagcatcctt gaacaaggac aattaacagt taacaaataa aaacgcaaaa gaaaatgccg
     6901 atatcctatt ggcattgacg tcaggtggca cttttcgagg agtttactag taagtaaagg
     6961 tggatacgga tccgaattcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc
     7021 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     7081 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     7141 ttttgctgaa aggaggaact atatccggat
//
