LOCUS       sequence-308652-        6530 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses in BL-21 (DE3) cells to produce the poly(aspartic acid) 
            hydrolase-1 from Sphingomonas Sp. KT-1 which selectively cleaves the
            beta linkages in thermally synthesized poly(aspartic acid)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6530)
  AUTHORS   Bolay A,  Hiester W, Davis N, Weiland M
  TITLE     Gel Assay to Introduce Polymer Biodegradation in the Undergraduate 
            Laboratory
  JOURNAL   Journal of Chemical Education
REFERENCE   2  (bases 1 to 6530)
  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: "Journal of 
            Chemical Education"
COMMENT     Sequence Label: pMW-PahZ1 KT-1
FEATURES             Location/Qualifiers
     source          1..6530
                     /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    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1031..1049)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1045..1092
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1267..1272,1273..1289,1290..1295))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1322..1340)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1408..1512
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1513..1581,1582..2373)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1731..1750)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2544..3132
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3033..3052
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3286..3303
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3318..3457
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3383..3402)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3543..3565
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3559..3750)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5062..6144)
                     /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     6106..6125
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6145..6222)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6428..6447
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg tcgaagggca aggcagccgc cttgcccgac
      181 ctcaaacccg gcgcgggcag cttcctgttc accggctggg cgggcaagcc gctgaaggtt
      241 cattattacg cgcccgacaa gatcacggaa acgacgcgca tattgttcgt catccacggc
      301 gccgggcgca acgccgacgg ctatcgggat gcgtggattc cctatgccaa agagggtcag
      361 tatatcgttc tgacccccga atattcgatg gccgatttcc cgacatcgct gacctataac
      421 gtcgggcata tcgtcgacga ggccggcaat ccgcggccgc gcgaggaatg gtcgttcgcg
      481 tcgatcgaac cgatgttcga ccaggtgcgc aaggcaacgg gcagcaaggt gccgacctat
      541 gcgatctatg gccattcggc gggcggtcag ttcgtccatc gctttgtcga gctgtggccc
      601 gatgcgcgct atagccgtgc cgttgcggcc aatgcgggct ggtacacgat gcccgacctc
      661 gcgatcaaat atccctatgg ccttaaagac gcgccgaccg atgcggcggg actgaaagcg
      721 acgttggaaa agccgctgac gatcctgctc ggcaccgcgg acaccgacgt caaccatcac
      781 cagctttcgc ggacgcccga agcgatgacc cagggtgtcc accggctggc gcgcggcgag
      841 ttcttctatg cctatggccg caaggtcgcg catgaactca acgccaaatt cgcatggaag
      901 ctcgattatg cgcccgacat tgcacacagc aatacgggca tgagccaata tgcccagaag
      961 cttgtctggg aataactcga ggatccggct gctaacaaag cccgaaagga agctgagttg
     1021 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1081 aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg cccggcagta
     1141 ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg atgacgatga
     1201 gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac tgtgataaac
     1261 taccgcatta aagcttatcg atgataagct gtcaaacatg agaattcttg aagacgaaag
     1321 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     1381 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     1441 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     1501 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     1561 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     1621 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     1681 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     1741 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     1801 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     1861 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     1921 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     1981 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     2041 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     2101 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     2161 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     2221 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     2281 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     2341 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     2401 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     2461 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2521 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2581 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2641 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     2701 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2761 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2821 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2881 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2941 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3001 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3061 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3121 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3181 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3241 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3301 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3361 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     3421 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     3481 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3541 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     3601 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     3661 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     3721 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     3781 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     3841 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     3901 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     3961 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     4021 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     4081 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     4141 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     4201 gatcatgcgc acccgtggcc aggacccaac gctgcccgag atgcgccgcg tgcggctgct
     4261 ggagatggcg gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct
     4321 ccgcaagaat tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg
     4381 gcttccattc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag
     4441 acaaggtata gggcggcgcc tacaatccat gccaacccgt tccatgtgct cgccgaggcg
     4501 gcataaatcg ccgtgacgat cagcggtcca atgatcgaag ttaggctggt aagagccgcg
     4561 agcgatcctt gaagctgtcc ctgatggtcg tcatctacct gcctggacag catggcctgc
     4621 aacgcgggca tcccgatgcc gccggaagcg agaagaatca taatggggaa ggccatccag
     4681 cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt cggccgccat gccggcgata
     4741 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4801 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     4861 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     4921 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     4981 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     5041 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5101 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     5161 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     5221 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     5281 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     5341 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5401 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5461 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5521 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5581 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5641 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5701 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5761 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5821 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5881 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5941 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     6001 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     6061 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     6121 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     6181 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     6241 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     6301 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6361 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     6421 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     6481 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
//
