LOCUS       sequence-220165-        4400 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 4400)
  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 4400)
  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..4400
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     40..59
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        40..58
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    58..82
                     /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             95..117
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             158..175
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(189..207)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(190..207)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(265..283)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      279..326
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(562..583)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(733..1815)
                     /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     1777..1796
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1816..1893)
                     /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     2036..2055
                     /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
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2797..2816)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     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 ctctccccgc gcgttggccg attcattaat gcagctgatt aatacgactc actataggga
       61 attgtgagcg gataacaatt cctctagaaa taattttgtt taactttaag aaggagatat
      121 atccatggat atcgaattcg tcgacctcga gggatcccac catcaccatc accattaagc
      181 ttgagtattc tatagtgtca cctaaatccc agcttgatcc ggctgctaac aaagcccgaa
      241 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
      301 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga taacctggcg
      361 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
      421 atggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
      481 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
      541 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
      601 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccacagct tgcatgcctg
      661 caggtcggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta acttacatta
      721 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
      781 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
      841 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
      901 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
      961 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     1021 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     1081 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     1141 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     1201 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     1261 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     1321 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     1381 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     1441 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     1501 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     1561 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     1621 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     1681 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     1741 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     1801 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     1861 accgcgaaag gttttgcacc attcgatggt gtcgggtacc gagctcgaat tgtgaaccat
     1921 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     1981 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2041 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2101 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcctgatgc 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 tgtggcgcgg tattatcccg
     2821 tattgacgcc gggcaagagc aactcggtcg ccgcatacac 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 tactgttctt 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
//
