LOCUS       sequence-368368-        6737 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Engineered Human Heparanase mutant for e.coli expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6737)
  AUTHORS   Whitefield C, Hong N, Mitchell JA, Jackson CJ
  TITLE     Computational design and experimental characterisation of a stable 
            human heparanase variant.
  JOURNAL   RSC Chem Biol. 2022 Feb 15;3(3):341-349. doi: 10.1039/d1cb00239b. 
            eCollection 2022 Mar 9.
  PUBMED    35382258
REFERENCE   2  (bases 1 to 6737)
  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"; doi: 
            "10.1039/d1cb00239b"; journalName: "RSC Chem Biol"; date: 
            "2022-02-15- 15"; volume: "3"; issue: "3"; pages: "341-349"
COMMENT     Sequence Label: pHPSE-P6
FEATURES             Location/Qualifiers
     source          1..6737
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        421..439
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    440..464
                     /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)."
     CDS             1686..1730
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1768..1786)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1782..1829
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1866..2321
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1953..1972)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2163..2184
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2436..3227,3228..3296))
                     /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     3059..3078
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3297..3389)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3470..4058
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3959..3978
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4212..4229
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4244..4383
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4309..4328)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4469..4491
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4485..4676)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5248..6330)
                     /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     6292..6311
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6331..6408)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6614..6633
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6721..6737,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcgcaggat gtggtcgatc ttgatttctt cacgcaggag cctttgcacc ttgtatcccc
      181 ctcattttta agcgttacca tagacgcaaa ccttgccact gacccgcgct tcttaatctt
      241 gcttggtagt ccaaagttaa gaacgctggc gcgggggctt agtccagcat atctgcgctt
      301 tggcggaacg aaaacagact tcctgatttt tgatcctaaa aaggaataaa agcttgcggc
      361 cgcataatgc ttaagtcgaa cagaaagtaa tcgtattgta cacggccgca taatcgaaat
      421 taatacgact cactataggg gaattgtgag cggataacaa ttccccatct tagtatatta
      481 gttaagtata agaaggagat atacatatga aaaaattcaa aaactcgacg tatagccggt
      541 cttctgtgga tgtgctctat acttttgcga agtgttcggg cctggactta atcttcggct
      601 taaatgcact gcttcggact tcagatgggc agtggaattc tagcaatgct cagctcctgc
      661 tcgattactg tgcctctaaa gggtataaca tcgactggga gttgggcaac gagccaaata
      721 gcttccgtaa aaaggctggg atcttcatca acgggtcgca attaggcaag gacttcattc
      781 accttcacaa actgctccgg aaatcgacat ttaagaatgc gaaactgtat ggccctgatg
      841 taggtcaacc gcgcgggaaa acggccaaaa tgcttaaatc gttcctgaag gcgggcggcg
      901 aagtcattga tgcagtaaca tggcaccatt actatttgaa tggtcgcacc gccaccttag
      961 aagatttcct gaatccggac gtattggaca cgtttatttc tcaggttcaa aaggtcttgc
     1021 aagttgtcga atcgacccgg cctgggaaga aagtttggct cggggagaca agttccgcct
     1081 atggcggtgg cgctcctggc ctctcagata cctttgctgc tggtttcatg tggcttgata
     1141 aactgggcct ctccgctcgc atggggatcg aagtcgtgat gcgccaagta ttttttggcg
     1201 ctggcaacta ccacctcgtc gacgaaaact tcgatccatt gcctgactac tggctgagcc
     1261 tccttttcaa aaagttagtt ggtacaaagg tgttgatggc aagtgttcag ggtcaggatc
     1321 gccgcaaact tcgcgtttat ctccattgca caaatacgga taatcctcgc tacaaagaag
     1381 gcgacctgac gctctatgct atcaacctcc ataacgtcac caagtatctc cgcctgccat
     1441 atccttttag taataaacag gtggatcaat atctcttgcg ccctcatggc cctgatggtt
     1501 tactgtccaa gagcgtgcag ttgaatggcc agaccctcaa gatggttgat gaccagactt
     1561 tgcctccttt gaagccaaaa ccactgcgtc cggggagcag tcttggcctg cctgccttct
     1621 cctacgcatt ttttgtaatt cgtaacgcaa aggtcccagc ctgcatctga taactcgagt
     1681 ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg
     1741 cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1801 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     1861 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1921 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1981 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2041 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2101 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2161 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2221 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2281 atttaacgcg aattttaaca aaatattaac gtttacaatt tctggcggca cgatggcatg
     2341 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2401 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2461 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     2521 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     2581 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     2641 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     2701 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     2761 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     2821 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     2881 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     2941 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3001 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3061 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3121 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3181 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3241 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3301 ttcctttttc aatcatgatt gaagcattta tcagggttat tgtctcatga gcggatacat
     3361 atttgaatgt atttagaaaa ataaacaaat aggtcatgac caaaatccct taacgtgagt
     3421 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3481 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3541 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3601 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3661 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3721 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3781 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3841 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3901 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3961 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4021 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4081 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4141 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4201 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     4261 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     4321 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     4381 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4441 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4501 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     4561 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     4621 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     4681 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     4741 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     4801 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     4861 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     4921 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     4981 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     5041 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     5101 agccgggtcc tcaacgacag gagcacgatc atgctagtca tgccccgcgc ccaccggaag
     5161 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     5221 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5281 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5341 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     5401 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     5461 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     5521 taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     5581 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     5641 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     5701 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     5761 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     5821 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     5881 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     5941 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     6001 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     6061 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     6121 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     6181 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     6241 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     6301 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     6361 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     6421 gctttccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     6481 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     6541 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     6601 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     6661 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat
     6721 taatacgact cactata
//
