LOCUS       sequence-212051-        4408 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as non-cleavable 
            C-terminal 6His-tag fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4408)
  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 4408)
  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: pHAT5
FEATURES             Location/Qualifiers
     source          1..4408
                     /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             119..136
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(150..168)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(151..168)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(226..244)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      240..287
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(523..544)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    663..684
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(697..1779)
                     /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     1741..1760
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1780..1857)
                     /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     2003..2022
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2178..2197)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2297..2319
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2357..2375)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2443..2547
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2548..2616,2617..3408)
                     /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      3579..4167
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4068..4087
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4321..4338
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgga tatcgaattc gtcgacctcg agggatccca
      121 ccatcaccat caccattaag cttgagtatt ctatagtgtc acctaaatcc cagcttgatc
      181 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
      241 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
      301 ctatatccgg ataacctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
      361 gttgcgcagc ctgaatggcg aatggaaatt gtaaacgtta atattttgtt aaaattcgcg
      421 ttaaatattt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
      481 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
      541 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
      601 ggcccactac agcttgcatg cctgcaggtc ggaagcataa agtgtaaagc ctggggtgcc
      661 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
      721 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
      781 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
      841 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
      901 aaaatcctgt ttgatggtgg ttgacggcgg gatataacat gagctgtctt cggtatcgtc
      961 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     1021 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     1081 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     1141 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     1201 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     1261 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     1321 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     1381 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     1441 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     1501 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     1561 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     1621 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     1681 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     1741 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     1801 ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcga tggtgtcggg
     1861 taccgagctc gaatttacgt gaaccatcac ccaaatcaag ttttttgcgg tcgaggtgcc
     1921 gtaaagctct aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     1981 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     2041 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     2101 agggcgcgtc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2161 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     2221 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     2281 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     2341 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     2401 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2461 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     2521 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     2581 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     2641 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     2701 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     2761 gttctgctat gtcatacact attatcccgt attgacgccg ggcaagagca actcggtcgc
     2821 cgggcgcggt attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     2881 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     2941 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     3001 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     3061 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     3121 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     3181 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     3241 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     3301 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     3361 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     3421 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     3481 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3541 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3601 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3661 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3721 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3781 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3841 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3901 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3961 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4021 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4081 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4141 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4201 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4261 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4321 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     4381 cgttggccga ttcattaatg cagctgat
//
