LOCUS       sequence-212048-        4443 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 4443)
  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 4443)
  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..4443
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    19..43
                     /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             56..78
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(185..203)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(186..203)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(261..279)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      275..322
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(558..579)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    698..719
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(732..1814)
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     1776..1795
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1815..1892)
                     /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."
     primer_bind     2038..2057
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2213..2232)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2332..2354
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2392..2410)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2478..2582
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2583..2651,2652..3443)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCHTLLSRIDAGQEQLGRRARYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3614..4202
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4103..4122
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4356..4373
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgag tcatcaccat caccatcact ccatggatat
      121 cgaattcgtc gacctcgagg gatccgggcc ctctagatgc ggccgcatgc ataagcttga
      181 gtattctata gtgtcaccta aatcccagct tgatccggct gctaacaaag cccgaaagga
      241 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
      301 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggataac ctggcgtaat
      361 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
      421 aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tatttgttaa atcagctcat
      481 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
      541 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      601 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacagctt gcatgcctgc
      661 aggtcggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
      721 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
      781 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
      841 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
      901 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttgac
      961 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     1021 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     1081 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     1141 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     1201 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     1261 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     1321 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     1381 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     1441 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     1501 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     1561 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     1621 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     1681 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     1741 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     1801 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     1861 ccgcgaaagg ttttgcacca ttcgatggtg tcgggtaccg agctcgaatt tacgtgaacc
     1921 atcacccaaa tcaagttttt tgcggtcgag gtgccgtaaa gctctaaatc ggaaccctaa
     1981 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     2041 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     2101 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcgcctg atgcggtatt
     2161 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct
     2221 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     2281 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     2341 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     2401 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     2461 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2521 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2581 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     2641 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     2701 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     2761 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtcat acactattat
     2821 cccgtattga cgccgggcaa gagcaactcg gtcgccgggc gcggtattct cagaatgact
     2881 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     2941 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     3001 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     3061 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     3121 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3181 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3241 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     3301 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3361 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     3421 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3481 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3541 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3601 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3661 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3721 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     3781 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3841 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3901 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3961 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4021 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4081 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4141 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4201 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4261 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4321 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4381 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     4441 gat
//
