LOCUS       sequence-212049-        4497 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 4497)
  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 4497)
  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..4497
                     /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"
     promoter        complement(239..257)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(240..257)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(315..333)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      329..376
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(612..633)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    752..773
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(786..1868)
                     /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     1830..1849
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1869..1946)
                     /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     2092..2111
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2267..2286)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2386..2408
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2446..2464)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2532..2636
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2637..2705,2706..3497)
                     /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      3668..4256
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4157..4176
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4410..4427
                     /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 ggatccatgg atatcgaatt
      181 cgtcgacctc gagggatccg ggccctctag atgcggccgc atgcataagc ttgagtattc
      241 tatagtgtca cctaaatccc agcttgatcc ggctgctaac aaagcccgaa aggaagctga
      301 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
      361 cttgaggggt tttttgctga aaggaggaac tatatccgga taacctggcg taatagcgaa
      421 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggaaattg
      481 taaacgttaa tattttgtta aaattcgcgt taaatatttg ttaaatcagc tcatttttta
      541 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
      601 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
      661 aagggcgaaa aaccgtctat cagggcgatg gcccactaca gcttgcatgc ctgcaggtcg
      721 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
      781 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
      841 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
      901 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
      961 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt tgacggcggg
     1021 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     1081 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     1141 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     1201 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     1261 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     1321 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     1381 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     1441 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     1501 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     1561 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     1621 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     1681 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     1741 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     1801 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     1861 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     1921 aaggttttgc accattcgat ggtgtcgggt accgagctcg aatttacgtg aaccatcacc
     1981 caaatcaagt tttttgcggt cgaggtgccg taaagctcta aatcggaacc ctaaagggag
     2041 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     2101 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     2161 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg cctgatgcgg tattttctcc
     2221 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg
     2281 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     2341 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     2401 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     2461 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     2521 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2581 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     2641 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     2701 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     2761 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     2821 aacgttttcc aatgatgagc acttttaaag ttctgctatg tcatacacta ttatcccgta
     2881 ttgacgccgg gcaagagcaa ctcggtcgcc gggcgcggta ttctcagaat gacttggttg
     2941 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3001 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     3061 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     3121 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     3181 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     3241 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     3301 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     3361 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     3421 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     3481 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     3541 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     3601 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3661 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3721 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3781 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     3841 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3901 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3961 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4021 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4081 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4141 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4201 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4261 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4321 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     4381 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     4441 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctgat
//
