LOCUS       sequence-174171-        5986 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG-inducible expression of T4 DNA ligase for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5986)
  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 5986)
  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-ligase
FEATURES             Location/Qualifiers
     source          1..5986
                     /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      1752..1846
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1916..1938)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        2402..2504
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             2505..3164
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2571..2590)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      3715..4303
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4204..4223
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4457..4474
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4547..4624
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4625..5707
                     /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(4644..4663)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5756..5761,5762..5779,5780..5786)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5794..5810
                     /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     5818..5834
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5818..5834
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5850..5867)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5850..5866)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5859..5881)
                     /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 gggccattct
      241 taaaattctg aacgaaatag catctattgg ttcaactaaa cagaagcaag caattcttga
      301 aaagaataaa gataatgaat tgcttaaacg agtatatcgt ctgacttatt ctcgtgggtt
      361 acagtattat atcaagaaat ggcctaaacc tggtattgct acccagagtt ttggaatgtt
      421 gactcttacc gatatgcttg acttcattga attcacatta gctactcgga aattgactgg
      481 aaatgcagca attgaggaat taactggata tatcaccgat ggtaaaaaag atgatgttga
      541 agttttgcgt cgagtgatga tgcgagacct tgaatgtggt gcttcagtat ctattgcaaa
      601 caaagtttgg ccaggtttaa ttcctgaaca acctcaaatg ctcgcaagtt cttatgatga
      661 aaaaggcatt aataagaata tcaaatttcc agcctttgct cagttaaaag ctgatggagc
      721 tcggtgtttt gctgaagtta gaggtgatga attagatgat gttcgtcttt tatcacgagc
      781 tggtaatgaa tatctaggat tagatcttct taaggaagag ttaattaaaa tgaccgctga
      841 agcccgccag attcatccag aaggtgtgtt gattgatggc gaattggtat accatgagca
      901 agttaaaaag gagccagaag gcctagattt tctttttgat gcttatcctg aaaacagtaa
      961 agctaaagaa ttcgccgaag ttgctgaatc acgtactgct tctaatggaa tcgccaataa
     1021 atctttaaag ggaaccattt ctgaaaaaga agcacaatgc atgaagtttc aggtctggga
     1081 ttatgtcccg ttggtagaaa tatacagtct tcctgcattt cgtttgaaat atgatgtacg
     1141 tttttctaaa ctagaacaaa tgacatctgg atatgataaa gtaattttaa ttgaaaacca
     1201 ggtagtaaat aacctagatg aagctaaggt aatttataaa aagtatattg accaaggtct
     1261 tgaaggtatt attctcaaaa atatcgatgg attatgggaa aatgctcgtt caaaaaatct
     1321 ttataaattt aaagaagtaa ttgatgttga tttaaaaatt gtaggaattt atcctcaccg
     1381 taaagaccct actaaagcgg gtggatttat tcttgagtca gagtgtggaa aaattaaggt
     1441 aaatgctggt tcaggcttaa aagataaagc cggtgtaaaa tcgcatgaac ttgaccgtac
     1501 tcgcattatg gaaaaccaaa attattatat tggaaaaatt ctagagtgcg aatgcaacgg
     1561 ttggttaaaa tctgatggcc gcactgatta cgttaaatta tttcttccga ttgcgattcg
     1621 tttacgtgaa gataaaacta aagctaatac attcgaagat gtatttggtg attttcatga
     1681 ggtaactggt ctataaggcc tatgcggccg ctaagggtcg acctgcagcc aagcttaatt
     1741 agctgagctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt
     1801 gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gctaggggaa
     1861 ttcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     1921 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     1981 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     2041 cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg atacgcctat
     2101 ttttataggt taatgtcatg ataataatag tttcttagaa tcctgatgtc cggcggtgct
     2161 tttgccgtta cgcaccaccc cgtcagtagc tgaacaggag ggacagctga tagaaacaga
     2221 agccactgga gcacctcaaa aacaccatca tacactaaat cagtaagttg gcagcatcac
     2281 ccgacgcact ttgcgccgaa taaatacctg tgacggaaga tcacttcgca gaataaataa
     2341 atcctggtgt ccctgttgat accgggaagc cctgggccaa cttttggcga aaatgagacg
     2401 ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg
     2461 tattttttga gttatcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca
     2521 ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc
     2581 agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa
     2641 agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc
     2701 tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg
     2761 atagtgttca cccttgttat accgttttcc atgagcaaac tgaaacgttt tcatcgctct
     2821 ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt
     2881 gttacggtga gaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct
     2941 cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact
     3001 tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc
     3061 cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta
     3121 atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt
     3181 attggtgccc ttaaacgcct ggttgctacg cctgaataag tgataataag cggatgaatg
     3241 gcagaaattc gaaagcaaat tcgacccggt cgtcggttca gggcagggtc gttaaatagc
     3301 cgcttatgtc tattgctggt ttaccggttt attgactacc ggaagcagtg tgaccgtgtg
     3361 cttctcaaat gcctgaggcc agtttgctca ggctctcccc gtggaggtaa taattgacga
     3421 tatgatcatt tattctgcct cccagagcct gataaaaacg gttaccggat cgagtcaggc
     3481 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3541 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3601 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3661 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3721 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3781 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3841 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     3901 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3961 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4021 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4081 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4141 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4201 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4261 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4321 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4381 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4441 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     4501 ttttctcctt acgcatctgt gcggtatttc acaccgcata aattccgaca ccatcgaatg
     4561 gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     4621 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     4681 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     4741 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     4801 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     4861 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     4921 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     4981 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     5041 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     5101 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     5161 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     5221 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     5281 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     5341 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     5401 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
     5461 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     5521 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     5581 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     5641 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     5701 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccc caggctttac
     5761 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     5821 gaaacagcta tgaccatgat tacggattca ctggccgtcg ttttacaacg tcgtgactgg
     5881 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     5941 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgc
//
