LOCUS       sequence-220159-        4435 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as non-cleavable minimal 
            N-terminal 6His-tag fusions .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4435)
  AUTHORS   Peranen J, Rikkonen M, Hyvonen M, Kaariainen L
  TITLE     T7 vectors with modified T7lac promoter for expression of proteins 
            in Escherichia coli.
  JOURNAL   Anal Biochem. 1996 May 1;236(2):371-3.
  PUBMED    8660525
REFERENCE   2  (bases 1 to 4435)
  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: "Anal 
            Biochem."; date: "1996-05-1"; volume: "236(2)"; pages: "371-3"
COMMENT     Sequence Label: pHAT2
FEATURES             Location/Qualifiers
     source          1..4435
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(96..143)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     139..157
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     promoter        215..233
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     215..232
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(309..326)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             340..362
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    375..399
                     /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(398..417)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(399..417)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(488..505)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(659..1247)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(739..758)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1418..2209,2210..2278))
                     /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     2041..2060
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2279..2383)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2451..2469
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2507..2529)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2629..2648
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2802..2821)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2964..3041
                     /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."
     CDS             3042..4124
                     /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(3061..3080)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     primer_bind     4274..4295
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
       61 acgccaggtt atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta
      121 gaggccccaa ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc
      181 tttcgggctt tgttagcagc cggatcaagc tgggatttag gtgacactat agaatactca
      241 agcttatgca tgcggccgca tctagagggc ccggatccct cgaggtcgac gaattcgata
      301 tccatggagt gatggtgatg gtgatgactc atggatatat ctccttctta aagttaaaca
      361 aaattatttc tagaggaatt gttatccgct cacaattccc tatagtgagt cgtattaatc
      421 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
      481 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
      541 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
      601 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
      661 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
      721 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
      781 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
      841 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
      901 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
      961 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     1021 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     1081 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     1141 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     1201 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     1261 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     1321 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     1381 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     1441 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     1501 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     1561 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     1621 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     1681 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     1741 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     1801 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     1861 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     1921 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     1981 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     2041 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     2101 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     2161 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     2221 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     2281 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     2341 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     2401 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     2461 tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     2521 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     2581 agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc
     2641 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     2701 aataccgcat caggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2761 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2821 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2881 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2941 ttcacaattc gagctcggta cccgacacca tcgaatggtg caaaaccttt cgcggtatgg
     3001 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     3061 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     3121 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     3181 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     3241 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     3301 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     3361 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     3421 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     3481 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     3541 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     3601 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     3661 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     3721 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     3781 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     3841 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     3901 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     3961 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     4021 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     4081 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     4141 gtaagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc gacctgcagg
     4201 catgcaagct gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4261 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     4321 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     4381 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttcca
//
