LOCUS       sequence-174180-        6202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG-inducible expression of sso7d-ligase fusion for protein 
            purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6202)
  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 6202)
  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-sso7d-ligase
FEATURES             Location/Qualifiers
     source          1..6202
                     /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      1968..2062
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2132..2154)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        2618..2720
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             2721..3380
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2787..2806)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      3931..4519
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4420..4439
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4673..4690
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4763..4840
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4841..5923
                     /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(4860..4879)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5972..5977,5978..5995,5996..6002)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6010..6026
                     /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     6034..6050
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6034..6050
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6066..6083)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6066..6082)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6075..6097)
                     /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 gggcgactgt
      241 taaattcaaa tacaaaggtg aggagaaaga agttgacatt tccaagatca aaaaggtatg
      301 gcgcgtgggc aaaatgatct ctttcactta cgacgaaggc ggcggtaaaa ccggtcgtgg
      361 cgcggtgagc gaaaaggacg cgccaaaaga actgctgcag atgctggaaa aacagaagaa
      421 aggcactagt ggcggaggct ccggcggtgg cattcttaaa attctgaacg aaatagcatc
      481 tattggttca actaaacaga agcaagcaat tcttgaaaag aataaagata atgaattgct
      541 taaacgagta tattgtctga cttattctcg tgggttacag tattatatca agaaatggcc
      601 taaacctggt attgctaccc agagttttgg aatgttgact cttaccgata tgcttgactt
      661 cattgaattc acattagcta ctcggaaatt gactggaaat gcagcaattg aggaattaac
      721 tggatatatc accgatggta aaaaagatga tgttgaagtt ttgcgtcgag tgatgatgcg
      781 agaccttgaa tgtggtgctt cagtatctat tgcaaacaaa gtttggccag gtttaattcc
      841 tgaacaacct caaatgctcg caagttctta tgatgaaaaa ggcattaata agaatatcaa
      901 atttccagcc tttgctcagt taaaagctga tggagctcgg tgttttgctg aagttagagg
      961 tgatgaatta gatgatgttc gtcttttatc acgagctggt aatgaatatc taggattaga
     1021 tcttcttaag gaagagttaa ttaaaatgac cgctgaagcc cgccagattc atccagaagg
     1081 tgtgttgatt gatggcgaat tggtatacca tgagcaagtt aaaaaggagc cagaaggcct
     1141 agattttctt tttgatgctt atcctgaaaa cagtaaagct aaagaattcg ccgaagttgc
     1201 tgaatcacgt actgcttcta atggaatcgc caataaatct ttaaagggaa ccatttctga
     1261 aaaagaagca caatgcatga agtttcaggt ctgggattat gtcccgttgg tagaaatata
     1321 cagtcttcct gcatttcgtt tgaaatatga tgtacgtttt tctaaactag aacaaatgac
     1381 atctggatat gataaagtaa ttttaattga aaaccaggta gtaaataacc tagatgaagc
     1441 taaggtaatt tataaaaagt atattgacca aggtcttgaa ggtattattc tcaaaaatat
     1501 cgatggatta tgggaaaatg ctcgttcaaa aaatctttat aaatttaaag aagtaattga
     1561 tgttgattta aaaattgtag gaatttatcc tcaccgtaaa gaccctacta aagcgggtgg
     1621 atttattctt gagtcagagt gtggaaaaat taaggtaaat gctggttcag gcttaaaaga
     1681 taaagccggt gtaaaatcgc atgaacttga ccgtactcgc attatggaaa accaaaatta
     1741 ttatattgga aaaattctag agtgcgaatg caacggttgg ttaaaatctg atggccgcac
     1801 tgattacgtt aaattatttc ttccgattgc gattcgttta cgtgaagata aaactaaagc
     1861 taatacattc gaagatgtat ttggtgattt tcatgaggta actggtctat aaggcctatg
     1921 cggccgctaa gggtcgacct gcagccaagc ttaattagct gagcttggac tcctgttgat
     1981 agatccagta atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg
     2041 ggcgtttttt attggtgaga atccaagcta ggggaattca tcgtgactga ctgacgatct
     2101 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     2161 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     2221 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
     2281 attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     2341 taatagtttc ttagaatcct gatgtccggc ggtgcttttg ccgttacgca ccaccccgtc
     2401 agtagctgaa caggagggac agctgataga aacagaagcc actggagcac ctcaaaaaca
     2461 ccatcataca ctaaatcagt aagttggcag catcacccga cgcactttgc gccgaataaa
     2521 tacctgtgac ggaagatcac ttcgcagaat aaataaatcc tggtgtccct gttgataccg
     2581 ggaagccctg ggccaacttt tggcgaaaat gagacgttga tcggcacgta agaggttcca
     2641 actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt
     2701 caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat
     2761 cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata
     2821 accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca
     2881 agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattcc
     2941 gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct tgttataccg
     3001 ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc
     3061 ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgagaac ctggcctatt
     3121 tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca
     3181 ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg
     3241 gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg
     3301 ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg
     3361 agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa acgcctggtt
     3421 gctacgcctg aataagtgat aataagcgga tgaatggcag aaattcgaaa gcaaattcga
     3481 cccggtcgtc ggttcagggc agggtcgtta aatagccgct tatgtctatt gctggtttac
     3541 cggtttattg actaccggaa gcagtgtgac cgtgtgcttc tcaaatgcct gaggccagtt
     3601 tgctcaggct ctccccgtgg aggtaataat tgacgatatg atcatttatt ctgcctccca
     3661 gagcctgata aaaacggtta ccggatcgag tcaggcaact atggatgaac gaaatagaca
     3721 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     3781 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     3841 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3901 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3961 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4021 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     4081 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4141 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4201 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4261 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4321 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4381 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4441 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4501 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4561 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4621 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4681 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4741 tatttcacac cgcataaatt ccgacaccat cgaatggcgc aaaacctttc gcggtatggc
     4801 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     4861 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     4921 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     4981 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     5041 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     5101 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     5161 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     5221 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     5281 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     5341 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     5401 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     5461 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     5521 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     5581 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     5641 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     5701 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     5761 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     5821 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     5881 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     5941 taagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     6001 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     6061 gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     6121 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     6181 gatcgccctt cccaacagtt gc
//
