LOCUS       sequence-200650-        5341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of lambda exonuclease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5341)
  AUTHORS   Zhang J, McCabe KA, Bell CE
  TITLE     Crystal structures of lambda exonuclease in complex with DNA suggest
            an electrostatic ratchet mechanism for processivity.
  JOURNAL   Proc Natl Acad Sci U S A. 2011 Jul 19;108(29):11872-7. doi: 
            10.1073/pnas.1103467108. Epub 2011 Jul 5.
  PUBMED    21730170
REFERENCE   2  (bases 1 to 5341)
  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: 
            "10.1073/pnas.1103467108"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2011-07-19- 19"; volume: "108"; issue: "29"; pages: "11872-7"
COMMENT     Sequence Label: pET14b – lambda exonuclease
FEATURES             Location/Qualifiers
     source          1..5341
                     /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"
     RBS             51..73
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             119..136
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             140..820
                     /codon_start=1
                     /gene="exo"
                     /product="5' to 3' double-stranded DNA exonuclease in the 
                     lambda Red system"
                     /label=Exo
                     /note="creates 3' single-stranded DNA tails"
                     /translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     primer_bind     complement(871..889)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      885..932
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1298..1303,1304..1320,1321..1326))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1353..1371)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1439..1543
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1544..1612,1613..2404)
                     /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(1762..1781)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2575..3163
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3064..3083
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3317..3334
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3349..3488
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3414..3433)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3574..3596
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3590..3781)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5239..5258
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
       61 tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca gcagcggcct
      121 ggtgccgcgc ggcagccata tgacaccgga cattatcctg cagcgtaccg ggatcgatgt
      181 gagagctgtc gaacaggggg atgatgcgtg gcacaaatta cggctcggcg tcatcaccgc
      241 ttcagaagtt cacaacgtga tagcaaaacc ccgctccgga aagaagtggc ctgacatgaa
      301 aatgtcctac ttccacaccc tgcttgctga ggtttgcacc ggtgtggctc cggaagttaa
      361 cgctaaagca ctggcctggg gaaaacagta cgagaacgac gccagaaccc tgtttgaatt
      421 cacttccggc gtgaatgtta ctgaatcccc gatcatctat cgcgacgaaa gtatgcgtac
      481 cgcctgctct cccgatggtt tatgcagtga cggcaacggc cttgaactga aatgcccgtt
      541 tacctcccgg gatttcatga agttccggct cggtggtttc gaggccataa agtcagctta
      601 catggcccag gtgcagtaca gcatgtgggt gacgcgaaaa aatgcctggt actttgccaa
      661 ctatgacccg cgtatgaagc gtgaaggcct gcattatgtc gtgattgagc gggatgaaaa
      721 gtacatggcg agttttgacg agatcgtgcc ggagttcatc gaaaaaatgg acgaggcact
      781 ggctgaaatt ggttttgtat ttggggagca atggcgatga ggatccggct gctaacaaag
      841 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
      901 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc
      961 cacaggacgg gtgtggtcgc catgatcgcg tagtcgatag tggctccaag tagcgaagcg
     1021 agcaggactg ggcggcggcc aaagcggtcg gacagtgctc cgagaacggg tgcgcataga
     1081 aattgcatca acgcatatag cgctagcagc acgccatagt gactggcgat gctgtcggaa
     1141 tggacgatat cccgcaagag gcccggcagt accggcataa ccaagcctat gcctacagca
     1201 tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat acacggtgcc
     1261 tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc gatgataagc
     1321 tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt
     1381 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     1441 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1501 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     1561 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     1621 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     1681 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     1741 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca
     1801 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     1861 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     1921 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     1981 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     2041 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac
     2101 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     2161 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     2221 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     2281 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     2341 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     2401 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     2461 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     2521 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2581 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2641 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2701 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     2761 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2821 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2881 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2941 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3001 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3061 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3121 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3181 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3241 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3301 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     3361 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     3421 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     3481 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3541 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3601 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     3661 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     3721 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     3781 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     3841 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     3901 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     3961 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     4021 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     4081 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     4141 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     4201 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa
     4261 cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg gatatgttct
     4321 gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct ccaattcttg
     4381 gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct
     4441 ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca
     4501 tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc
     4561 aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc
     4621 gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc
     4681 gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta
     4741 gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     4801 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     4861 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4921 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4981 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     5041 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     5101 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     5161 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     5221 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     5281 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     5341 t
//
