LOCUS       sequence-478436-        5925 bp    DNA     circular SYN 08-AUG-2025
DEFINITION  Production of a thermostable enzyme, Pyrobaculum calidifontis 
            manganese-dependent catalase, to be used in an educational 
            laboratory exercise..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5925)
  TITLE     Overproduction of a recombinant heat stable catalase for use in an 
            extremophile enzyme activity lab.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5925)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 8, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAJC1
FEATURES             Location/Qualifiers
     source          1..5925
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    733..872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(798..817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     958..980
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(974..1165)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1737..2819)
                     /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     2781..2800
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2820..2897)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3103..3122
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3210..3229
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3210..3228
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3229..3253
                     /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             3268..3290
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4216..4239
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4302..4320)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4316..4363
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4400..4855
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4487..4506)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4697..4718
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4947..5762)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4947..5762)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5670..5689
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5754..5773
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(5884..5925,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
      841 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
      901 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      961 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     1021 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     1081 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     1141 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     1201 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     1261 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     1321 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     1381 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     1441 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     1501 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     1561 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     1621 tgctagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     1681 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     1741 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     1801 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     1861 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     1921 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     1981 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     2041 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     2101 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     2161 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     2221 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     2281 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     2341 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     2401 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     2461 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     2521 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     2581 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     2641 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     2701 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     2761 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     2821 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     2881 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     2941 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     3001 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     3061 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     3121 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     3181 gatcgagatc gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     3241 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg
     3301 tacctaagaa tagaccggct ccaaatacaa ctgccagctc caaaagagcc tgaccccaac
     3361 gccgcggcag ccgtcagcga gttgcttggg ggcagatttg gggaaatgaa cacgttgatg
     3421 acgtacactt accaatcctt caacttccgc atgcacaaaa acccagccat aaagcccttt
     3481 agagacctgg tctccaacat agccacagag gaactcggtc acatcgagct ggtatccgcc
     3541 gtggtaaacg ccctatatgt gggctccaca aagccggcgc ccccagacca agccccacta
     3601 aagcccctaa aagacgtgag aaacacctac cacgcgataa acacaggcct cggcgccttc
     3661 cccatggact cccacggaac gccgtggcgc ggcgactaca tattcgtaag cggcaaccta
     3721 gtgctagact tcctatacaa cttcttccta gaggttgggg cacgcctagc caaaatcagg
     3781 gtatacgaga tgacagacaa cccagtggcc cgggaaatga tcggctacct actggtgcgc
     3841 ggcggcgtac acgccatggc atacggcaaa gccctagagg ctctgacagg ggtagaagtg
     3901 tggagaatgc tacccatacc caagatagag gactccaaat tcccagaagc cgccaagtac
     3961 atgaaaatgg gagtacacag gacactgtac agatttagcc caagcgacta caaagacata
     4021 gaaaaaatct ggaagggagc ccacccagcc gacggccagc cgctacaagt acacgacgga
     4081 cctcccgagg gaggagaata cctagaacta ccagaagtcc ccgaggaatt cgcccccggc
     4141 ctatacaaag acgactttga gagaatcgcc aagagactgg gaataaacct ctaaaagctt
     4201 gcggccgcac tcgagcacca ccaccaccac caccaccact aattgattaa tacctaggct
     4261 gctaaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     4321 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     4381 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4441 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     4501 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4561 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     4621 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4681 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     4741 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     4801 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     4861 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4921 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     4981 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     5041 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     5101 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     5161 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     5221 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     5281 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta
     5341 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     5401 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     5461 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     5521 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     5581 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     5641 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     5701 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     5761 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat
     5821 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5881 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaac
//
