LOCUS       sequence-220153-        4431 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 4431)
  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 4431)
  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..4431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..67
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        48..66
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    66..90
                     /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             103..125
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             145..162
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(228..246)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(229..246)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(304..322)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      318..365
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(601..622)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(772..1854)
                     /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     1816..1835
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1855..1932)
                     /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     2075..2094
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2248..2267)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2367..2389
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2427..2445)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2513..2617
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2618..2686,2687..3478)
                     /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(2836..2855)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3649..4237
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4138..4157
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4391..4408
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctgattaa tacgactcac
       61 tatagggaat tgtgagcgga taacaattcc tctagaaata attttgttta actttaagaa
      121 ggagatatat ccatgaacac cattcatcac catcaccatc acaacactag tagcgctacc
      181 atggatccgg gccctctaga tgcggccgca tgcataagct tgagtattct atagtgtcac
      241 ctaaatccca gcttgatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
      301 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
      361 ttttgctgaa aggaggaact atatccggat aacctggcgt aatagcgaag aggcccgcac
      421 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt aagcgttaat
      481 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
      541 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
      601 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
      661 accgtctatc agggcgatgg cccacagctt gcatgcctgc aggtcggaag cataaagtgt
      721 aaagcctggg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
      781 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
      841 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
      901 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
      961 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     1021 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     1081 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     1141 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     1201 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     1261 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     1321 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     1381 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     1441 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     1501 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     1561 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     1621 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     1681 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     1741 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     1801 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     1861 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcacca
     1921 ttcgatggtg tcgggtaccg agctcgaatt gtgaaccatc accctaatca agttttttgg
     1981 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     2041 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     2101 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     2161 atgcgccgct acagggcgcg tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     2221 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     2281 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     2341 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     2401 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2461 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     2521 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2581 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     2641 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2701 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     2761 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     2821 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     2881 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     2941 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3001 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3061 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3121 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     3181 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3241 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3301 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     3361 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3421 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3481 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3541 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3601 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3661 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3721 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3781 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3841 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3901 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3961 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4021 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4081 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4141 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4201 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4261 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4321 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4381 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac g
//
