LOCUS       sequence-212047-        4439 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as non-cleavable 
            N-terminal 6His-tag fusions .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4439)
  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 4439)
  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: pHAT
FEATURES             Location/Qualifiers
     source          1..4439
                     /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             98..115
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(181..199)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(182..199)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(257..275)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      271..318
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(554..575)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    694..715
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(728..1810)
                     /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     1772..1791
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1811..1888)
                     /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     2034..2053
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2209..2228)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2328..2350
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2388..2406)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2474..2578
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2579..2647,2648..3439)
                     /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      3610..4198
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4099..4118
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4352..4369
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa caccattcat caccatcacc atcacaacac
      121 tagtagcgct accatggatc cgggccctct agatgcggcc gcatgcataa gcttgagtat
      181 tctatagtgt cacctaaatc ccagcttgat ccggctgcta acaaagcccg aaaggaagct
      241 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
      301 gtcttgaggg gttttttgct gaaaggagga actatatccg gataacctgg cgtaatagcg
      361 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggaaat
      421 tgtaaacgtt aatattttgt taaaattcgc gttaaatatt tgttaaatca gctcattttt
      481 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
      541 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
      601 caaagggcga aaaaccgtct atcagggcga tggcccacta cagcttgcat gcctgcaggt
      661 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
      721 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
      781 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
      841 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
      901 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttgacggcg
      961 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     1021 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     1081 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     1141 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     1201 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     1261 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     1321 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     1381 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     1441 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     1501 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     1561 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     1621 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     1681 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     1741 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     1801 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     1861 gaaaggtttt gcaccattcg atggtgtcgg gtaccgagct cgaatttacg tgaaccatca
     1921 cccaaatcaa gttttttgcg gtcgaggtgc cgtaaagctc taaatcggaa ccctaaaggg
     1981 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     2041 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     2101 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cgcctgatgc ggtattttct
     2161 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     2221 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     2281 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     2341 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     2401 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     2461 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2521 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     2581 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     2641 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     2701 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     2761 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtcatacac tattatcccg
     2821 tattgacgcc gggcaagagc aactcggtcg ccgggcgcgg tattctcaga atgacttggt
     2881 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     2941 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     3001 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     3061 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3121 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3181 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3241 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     3301 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3361 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3421 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     3481 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3541 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3601 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3661 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3721 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     3781 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3841 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3901 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3961 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4021 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4081 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4141 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4201 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4261 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4321 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4381 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctgat
//
