LOCUS       sequence-220265-        4519 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 4519)
  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 4519)
  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..4519
                     /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"
     CDS             complement(813..1895)
                     /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     1857..1876
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1896..1973)
                     /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     2116..2135
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2289..2308)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2408..2430
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2468..2486)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2554..2658
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2659..2727,2728..3519)
                     /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(2877..2896)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3690..4278
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4179..4198
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4432..4449
                     /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 taagcgttaa tattttgtta aaattcgcgt
      541 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
      601 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
      661 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
      721 gcccacagct tgcatgcctg caggtcggaa gcataaagtg taaagcctgg ggtgcctaat
      781 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
      841 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
      901 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
      961 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     1021 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     1081 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     1141 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     1201 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     1261 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     1321 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     1381 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     1441 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     1501 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     1561 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     1621 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     1681 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     1741 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     1801 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     1861 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     1921 tccgggcgct atcatgccat accgcgaaag gttttgcacc attcgatggt gtcgggtacc
     1981 gagctcgaat tgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     2041 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2101 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2161 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2221 gtcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc
     2281 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     2341 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     2401 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     2461 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     2521 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2581 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2641 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     2701 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     2761 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     2821 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2881 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     2941 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     3001 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     3061 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3121 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3181 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     3241 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3301 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3361 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     3421 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     3481 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     3541 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     3601 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3661 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3721 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3781 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     3841 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3901 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3961 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4021 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4081 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4141 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4201 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4261 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4321 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4381 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4441 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     4501 attcattaat gcagctgat
//
