LOCUS       sequence-220164-        4489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as Thrombin cleavable 
            N-terminal 6His-tag fusions .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4489)
  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 4489)
  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: pHAT3
FEATURES             Location/Qualifiers
     source          1..4489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     85..104
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        85..103
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    103..127
                     /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             140..162
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             182..199
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             233..250
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     promoter        complement(323..341)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(324..341)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(399..417)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      413..460
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(696..717)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(867..1949)
                     /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     1911..1930
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1950..2027)
                     /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     2170..2189
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2343..2362)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2462..2484
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2522..2540)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2608..2712
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2713..2781,2782..3573)
                     /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     complement(2931..2950)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3744..4332
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4233..4252
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(4486..4489,1..14)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
       61 ggccgattca ttaatgcagc tgattaatac gactcactat agggaattgt gagcggataa
      121 caattcctct agaaataatt ttgtttaact ttaagaagga gatatatcca tgaacaccat
      181 tcatcaccat caccatcaca acactagtgg atctggtggt ggtggcggcc gcctggttcc
      241 gaggggatcc atggatatcg aattcgtcga cctcgaggga tccgggccct ctagatgcgg
      301 ccgcatgcat aagcttgagt attctatagt gtcacctaaa tcccagcttg atccggctgc
      361 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
      421 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
      481 cggataacct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
      541 agcctgaatg gcgaatggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
      601 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
      661 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
      721 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
      781 agcttgcatg cctgcaggtc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
      841 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
      901 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
      961 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     1021 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     1081 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     1141 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     1201 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     1261 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     1321 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     1381 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     1441 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     1501 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     1561 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     1621 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     1681 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     1741 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     1801 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     1861 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     1921 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     1981 cgctatcatg ccataccgcg aaaggttttg caccattcga tggtgtcggg taccgagctc
     2041 gaattgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     2101 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     2161 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     2221 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcctg
     2281 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
     2341 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     2401 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     2461 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     2521 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     2581 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2641 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2701 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     2761 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     2821 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     2881 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     2941 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     3001 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     3061 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     3121 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     3181 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     3241 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     3301 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     3361 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     3421 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     3481 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     3541 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     3601 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3661 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3721 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3781 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3841 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     3901 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3961 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4021 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4081 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4141 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4201 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4261 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4321 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4381 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4441 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcg
//
