LOCUS       sequence-171102-        7004 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG-inducible expression of p50-ligase fusion for protein 
            purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7004)
  AUTHORS   Wilson RH, Morton SK, Deiderick H, Gerth ML, Paul HA, Gerber I, 
            Patel A, Ellington AD, Hunicke-Smith SP, Patrick WM
  TITLE     Engineered DNA ligases with improved activities in vitro.
  JOURNAL   Protein Eng Des Sel. 2013 Jul;26(7):471-8. doi: 
            10.1093/protein/gzt024. Epub 2013 Jun 10.
  PUBMED    23754529
REFERENCE   2  (bases 1 to 7004)
  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: 
            "10.1093/protein/gzt024"; journalName: "Protein Eng Des Sel"; date: 
            "2013-07"; volume: "26"; issue: "7"; pages: "471-8"
COMMENT     Sequence Label: pCA24N-p50-ligase
FEATURES             Location/Qualifiers
     source          1..7004
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(80..94,95..100,101..117,118..124)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    100..116
                     /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)."
     protein_bind    132..148
                     /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             167..178
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             197..214
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2772..2866
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2936..2958)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3422..3524
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3525..4184
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3591..3610)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      4733..5321
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5222..5241
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5475..5492
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5565..5642
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             5643..6725
                     /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     complement(5662..5681)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    6738..6759
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6774..6779,6780..6797,6798..6804)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6812..6828
                     /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)."
     primer_bind     6836..6852
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6836..6852
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6868..6885)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6868..6884)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6877..6899)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
       61 tcgtcttcac ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat
      121 aatagattca attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt
      181 aactatgaga ggatctcacc atcaccatca ccatacggat ccggccctga gggccgcaga
      241 tggcccatac cttcaaatat tagagcaacc taaacagaga ggatttcgtt tccgttatgt
      301 atgtgaaggc ccatcccatg gtggattacc tggtgcctct agtgaaaaga acaagaagtc
      361 ttaccctcag gtcaaaatct gcaactatgt gggaccagca aaggttattg ttcagttggt
      421 cacaaatgga aaaaatatcc acctgcatgc ccacagcctg gtgggaaaac actgtgagga
      481 tgggatctgc actgtaactg ctggacccaa ggacatggtg gtcggcttcg caaacctggg
      541 tatacttcat gtgacaaaga aaaaagtatt tgaaacactg gaagcacgaa tgacagaggc
      601 gtgtataagg ggctataatc ctggactctt ggtgcaccct gaccttgcct atttgcaagc
      661 agaaggtgga ggggaccggc agctgggaga tcgggaaaaa gagctaatcc gccaagcagc
      721 tctgcagcag accaaggaga tggacctcag cgtggtgcgg ctcatgttta cagcttttct
      781 tccggatagc actggcagct tcacaaggcg cctggaaccc gtggtatcag acgccatcta
      841 tgacagtaaa gcccccaatg catccaactt gaaaattgta agaatggaca ggacagctgg
      901 atgtgtgact ggaggggagg aaatttatct tctttgtgac aaagttcaga aagatgacat
      961 ccagattcga ttttatgaag aggaagaaaa tggtggagtc tgggaaggat ttggagattt
     1021 ttcccccaca gatgttcata gacaatttgc cattgtcttc aaaactccaa agtataaaga
     1081 tattaatatt acaaaaccag cctctgtgtt tgtccagctt cggaggaaat ctgacttgga
     1141 aaccagtgaa ccaaaacctt tcctctacta tcctgaaatc aaagataaag aagaagtgca
     1201 gaggaaacgt cagaagggga gctcgggcac tagtggcgga ggctccggcg gtggcattct
     1261 taaaattctg aacgaaatag catctattgg ttcaactaaa cagaagcaag caattcttga
     1321 aaagaataaa gataatgaat tgcttaaacg agtatatcgt ctgacttatt ctcgtgggtt
     1381 acagtattat atcaagaaat ggcctaaacc tggtattgct acccagagtt ttggaatgtt
     1441 gactcttacc gatatgcttg acttcattga attcacatta gctactcgga aattgactgg
     1501 aaatgcagca attgaggaat taactggata tatcaccgat ggtaaaaaag atgatgttga
     1561 agttttgcgt cgagtgatga tgcgagacct tgaatgtggt gcttcagtat ctattgcaaa
     1621 caaagtttgg ccaggtttaa ttcctgaaca acctcaaatg ctcgcaagtt cttatgatga
     1681 aaaaggcatt aataagaata tcaaatttcc agcctttgct cagttaaaag ctgatggagc
     1741 tcggtgtttt gctgaagtta gaggtgatga attagatgat gttcgtcttt tatcacgagc
     1801 tggtaatgaa tatctaggat tagatcttct taaggaagag ttaattaaaa tgaccgctga
     1861 agcccgccag attcatccag aaggtgtgtt gattgatggc gaattggtat accatgagca
     1921 agttaaaaag gagccagaag gcctagattt tctttttgat gcttatcctg aaaacagtaa
     1981 agctaaagaa ttcgccgaag ttgctgaatc acgtactgct tctaatggaa tcgccaataa
     2041 atctttaaag ggaaccattt ctgaaaaaga agcacaatgc atgaagtttc aggtctggga
     2101 ttatgtcccg ttggtagaaa tatacagtct tcctgcattt cgtttgaaat atgatgtacg
     2161 tttttctaaa ctagaacaaa tgacatctgg atatgataaa gtaattttaa ttgaaaacca
     2221 ggtagtaaat aacctagatg aagctaaggt aatttataaa aagtatattg accaaggtct
     2281 tgaaggtatt attctcaaaa atatcgatgg attatgggaa aatgctcgtt caaaaaatct
     2341 ttataaattt aaagaagtaa ttgatgttga tttaaaaatt gtaggaattt atcctcaccg
     2401 taaagaccct actaaagcgg gtggatttat tcttgagtca gagtgtggaa aaattaaggt
     2461 aaatgctggt tcaggcttaa aagataaagc cggtgtaaaa tcgcatgaac ttgaccgtac
     2521 tcgcattatg gaaaaccaaa attattatat tggaaaaatt ctagagtgcg aatgcaacgg
     2581 ttggttaaaa tctgatggcc gcactgatta cgttaaatta tttcttccga ttgcgattcg
     2641 tttacgtgaa gataaaacta aagctaatac attcgaagat gtatttggtg attttcatga
     2701 ggtaactggt ctataaggcc tatgcggccg ctaagggtcg acctgcagcc aagcttaatt
     2761 agctgagctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt
     2821 gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gctaggggaa
     2881 ttcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     2941 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3001 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     3061 cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg atacgcctat
     3121 ttttataggt taatgtcatg ataataatgg tttcttagaa tcctgatgtc cggcggtgct
     3181 tttgccgtta cgcaccaccc cgtcagtagc tgaacaggag ggacagctga tagaaacaga
     3241 agccactgga gcacctcaaa aacaccatca tacactaaat cagtaagttg gcagcatcac
     3301 ccgacgcact ttgcgccgaa taaatacctg tgacggaaga tcacttcgca gaataaataa
     3361 atcctggtgt ccctgttgat accgggaagc cctgggccaa cttttggcga aaatgagacg
     3421 ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg
     3481 tattttttga gttatcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca
     3541 ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc
     3601 agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa
     3661 agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc
     3721 tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg
     3781 atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct
     3841 ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt
     3901 gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct
     3961 cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact
     4021 tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc
     4081 cgctggcgat tcaggttcat catgccgtct gtgatggctt ccatgtcggc agaatgctta
     4141 atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt
     4201 attggtgccc ttaaacgcct ggtgctacgc ctgaataagt gataataagc ggatgaatgg
     4261 cagaaattcg aaagcaaatt cgacccggtc gtcggttcag ggcagggtcg ttaaatagcc
     4321 gcttatgtct attgctggtt taccggttta ttgactaccg gaagcagtgt gaccgtgtgc
     4381 ttctcaaatg cctgaggcca gtttgctcag gctctccccg tggaggtaat aattgacgat
     4441 atgatcattt attctgcctc ccagagcctg ataaaaacgg ttaggatcgg agtcaggcaa
     4501 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     4561 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     4621 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     4681 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4741 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4801 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4861 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4921 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4981 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5041 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5101 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     5161 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     5221 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     5281 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     5341 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     5401 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     5461 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     5521 ttctccttac gcatctgtgc ggtatttcac accgcataaa ttccgacacc atcgaatggt
     5581 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     5641 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     5701 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     5761 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     5821 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     5881 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     5941 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     6001 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     6061 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     6121 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     6181 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     6241 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     6301 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     6361 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     6421 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     6481 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     6541 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     6601 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     6661 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     6721 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     6781 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     6841 aacagctatg accatgatta cggattcact ggccgtcgtt ttacaacgtc gtgactggga
     6901 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     6961 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgc
//
