LOCUS       sequence-212050-        4527 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as Thrombin and TEV 
            cleavable N-terminal 6His-tag fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4527)
  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 4527)
  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: pHAT4
FEATURES             Location/Qualifiers
     source          1..4527
                     /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"
     CDS             149..166
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             173..193
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(269..287)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(270..287)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(345..363)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      359..406
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(642..663)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    782..803
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(816..1898)
                     /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     1860..1879
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1899..1976)
                     /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     2122..2141
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2297..2316)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2416..2438
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2476..2494)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2562..2666
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2667..2735,2736..3527)
                     /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      3698..4286
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4187..4206
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4440..4457
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa caccattcat caccatcacc atcacaacac
      121 tagtggatct ggtggtggtg gcggccgcct ggttccgagg ggatccatgt ctgaaaacct
      181 gtacttccag ggttccatgg atatcgaatt cgtcgacctc gagggatccg ggccctctag
      241 atgcggccgc atgcataagc ttgagtattc tatagtgtca cctaaatccc agcttgatcc
      301 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
      361 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
      421 tatatccgga taacctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      481 ttgcgcagcc tgaatggcga atggaaattg taaacgttaa tattttgtta aaattcgcgt
      541 taaatatttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
      601 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
      661 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
      721 gcccactaca gcttgcatgc ctgcaggtcg gaagcataaa gtgtaaagcc tggggtgcct
      781 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
      841 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
      901 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
      961 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     1021 aaatcctgtt tgatggtggt tgacggcggg atataacatg agctgtcttc ggtatcgtcg
     1081 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     1141 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     1201 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     1261 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     1321 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     1381 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     1441 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     1501 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     1561 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     1621 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     1681 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     1741 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     1801 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     1861 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     1921 tcttccgggc gctatcatgc cataccgcga aaggttttgc accattcgat ggtgtcgggt
     1981 accgagctcg aatttacgtg aaccatcacc caaatcaagt tttttgcggt cgaggtgccg
     2041 taaagctcta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     2101 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     2161 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     2221 gggcgcgtcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2281 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     2341 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     2401 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     2461 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     2521 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2581 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2641 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     2701 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     2761 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2821 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     2881 ttctgctatg tcatacacta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     2941 gggcgcggta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3001 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3061 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3121 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3181 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3241 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3301 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3361 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     3421 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     3481 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     3541 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     3601 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3661 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3721 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3781 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3841 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3901 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3961 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4021 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4081 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4141 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4201 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4261 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4321 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4381 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4441 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     4501 gttggccgat tcattaatgc agctgat
//
