LOCUS       sequence-193367-        6885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of hydroxylated Hif1a TAD (776-826).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6885)
  AUTHORS   Sugase K, Landes MA, Wright PE, Martinez-Yamout M
  TITLE     Overexpression of post-translationally modified peptides in 
            Escherichia coli by co-expression with modifying enzymes.
  JOURNAL   Protein Expr Purif. 2008 Feb;57(2):108-15. Epub 2007 Nov 1.
  PUBMED    18054500
REFERENCE   2  (bases 1 to 6885)
  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: "Protein 
            Expr Purif."; date: "2008-02"; volume: "57(2)"; pages: "108-15. Epub
            2007 Nov 1"
COMMENT     Sequence Label: HisGB1-Hif1a(776-826)_FIH
FEATURES             Location/Qualifiers
     source          1..6885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      26..614
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     515..534
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     768..785
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    800..939
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(865..884)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1025..1047
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1041..1232)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2041..3123)
                     /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     3085..3104
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3124..3201)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3407..3426
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3510..3529
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3510..3528
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3529..3553
                     /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             3568..3590
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3603..3620
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3636..3797
                     /codon_start=1
                     /product="B1 domain of Streptococcal protein G   "
                     /label=GB1
                     /note="effective as a solubilizing fusion partner (Cheng 
                     and Patel, 2004)"
                     /translation="YKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
                     ATKTFTVTE"
     CDS             3822..3839
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     RBS             4025..4047
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      4052..4061
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5126..5143
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5196..5214)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5210..5257
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5294..5749
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5381..5400)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5591..5612
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5775..5879
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5880..5948,5949..6740)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6098..6117)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
       61 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      121 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
      181 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      241 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      301 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      361 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      421 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      481 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      541 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      601 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      661 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      721 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
      781 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
      841 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
      901 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
      961 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     1021 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     1081 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     1141 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     1201 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     1261 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     1321 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     1381 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     1441 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     1501 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     1561 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     1621 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     1681 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     1741 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     1801 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     1861 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     1921 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     1981 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     2041 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     2101 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     2161 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     2221 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     2281 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     2341 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     2401 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     2461 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     2521 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     2581 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     2641 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     2701 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     2761 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     2821 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     2881 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     2941 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     3001 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     3061 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     3121 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     3181 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     3241 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     3301 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     3361 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     3421 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     3481 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     3541 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg
     3601 gccatcatca tcatcatcac agcagcggca tgacttacaa actgatcctt aacggtaaaa
     3661 ctttgaaagg cgaaaccact accgaagctg ttgatgctgc gactgcagaa aaagttttca
     3721 aacagtacgc taacgacaac ggtgttgacg gtgaatggac ttacgacgat gcgactaaga
     3781 ccttcaccgt tactgaagaa ttcagcagcg gcagcagcgg cctggttccg cgtggttccc
     3841 atatgtctga tttagcatgt agactgctgg ggcaatcaat ggatgaaagt ggattaccac
     3901 agctgaccag ttatgattgt gaagttaatg ctcctataca aggcagcaga aacctactgc
     3961 agggtgaaga attactcaga gctttggatc aagttaactg atagggatcc cctctagaaa
     4021 taattttgtt taactttaag aaggagatat accatggcgg cgacagcggc ggaggctgtg
     4081 gcctctggct ctggagagcc ccgggaggag gctggagccc tcggccccgc ctgggatgaa
     4141 tcccagttgc gcagttatag cttcccgact aggcccattc cgcgtctgag tcagagcgac
     4201 ccccgggcag aggagcttat tgagaatgag gagcctgtgg tgctgaccga cacaaatctt
     4261 gtgtatcctg ccctgaaatg ggaccttgaa tacctgcaag agaatattgg caatggagac
     4321 ttctctgtgt acagtgccag cacccacaag ttcttgtact atgatgagaa gaagatggcc
     4381 aatttccaga actttaagcc gaggtccaac agggaagaaa tgaaatttca tgagttcgtt
     4441 gagaaactgc aggatataca gcagcgagga ggggaagaga ggttgtatct gcagcaaacg
     4501 ctcaatgaca ctgtgggcag gaagattgtc atggacttct taggttttaa ctggaactgg
     4561 attaataagc aacagggaaa gcgtggctgg gggcagctta cctctaacct gctgctcatt
     4621 ggcatggaag gaaatgtgac acctgctcac tatgatgagc agcagaactt ttttgctcag
     4681 ataaaaggtt acaaacgatg catcttattc cctccggatc agttcgagtg cctctaccca
     4741 taccctgttc atcacccatg tgacagacag agccaggtgg actttgacaa tcccgactac
     4801 gagaggttcc ctaatttcca aaatgtggtt ggttacgaaa cagtggttgg ccctggtgat
     4861 gttctttaca tcccaatgta ctggtggcat cacatagagt cattactaaa tggggggatt
     4921 accatcactg tgaacttctg gtataagggg gctcccaccc ctaagagaat tgaatatcct
     4981 ctcaaagctc atcagaaagt ggccataatg agaaacattg agaagatgct tggagaggcc
     5041 ttggggaacc cacaagaggt ggggcccttg ttgaacacaa tgatcaaggg ccgatacaac
     5101 tagtaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     5161 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     5221 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     5281 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     5341 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     5401 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     5461 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     5521 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5581 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5641 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5701 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     5761 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5821 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5881 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5941 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     6001 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     6061 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     6121 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     6181 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     6241 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     6301 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     6361 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     6421 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6481 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6541 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6601 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6661 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6721 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6781 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6841 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agacc
//
