LOCUS       sequence-174177-        6866 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG-inducible expression of ligase-pprA fusion for protein 
            purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6866)
  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 6866)
  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-pprA
FEATURES             Location/Qualifiers
     source          1..6866
                     /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      2632..2726
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2796..2818)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3282..3384
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3385..4044
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3451..3470)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      4595..5183
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5084..5103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5337..5354
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5427..5504
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5505..6587
                     /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(5524..5543)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6636..6641,6642..6659,6660..6666)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6674..6690
                     /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     6698..6714
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6698..6714
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6730..6747)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6730..6746)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6739..6761)
                     /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 ctaggcacta gtggcggagg ctccggcggt gcccgtgcca aggctaaaga
     1741 tcagacggac ggtatctacg cagcgttcga caccctgatg agcacggcgg gtgttgactc
     1801 tcagattgcg gcactggctg cgagcgaagc cgacgccggc accctggatg cggcgttgac
     1861 gcaatcgctg caagaggcgc agggccgctg gggtctgggc ctgcaccact tgcgccatga
     1921 ggcgcgtttg accgatgatg gtgacattga gatcctgacc gacggtcgcc ctagcgcacg
     1981 cgttagcgag ggttttggtg ccctggcgca agcctacgca ccgatgcaag cgctggatga
     2041 acgtggcctg tcccaatggg ctgcgctggg cgagggttat cgcgcaccgg gcgatctgcc
     2101 gctggctcag ctgaaggttc tgatcgaaca tgcccgcgat ttcgaaactg attggagcgc
     2161 gggtcgtggc gaaacgttcc aacgcgtctg gcgtaaaggt gacaccctgt ttgtcgaggt
     2221 ggcgcgtccg gcatccgccg aagcggcgct gagcgacgcg gcgtgggatg tgattgccag
     2281 catcaaggac cgtgcgttcc agcgtgagct gatgcgtcgt tctgagaaag acggcatgct
     2341 gggtgcgctg ttgggtgctc gccatgctgg cgccaaggca aacttggcac agctgccaga
     2401 ggcccacttt accgtgcagg catttgttca gactctgagc ggtgcggcag cacgtaatgc
     2461 ggaagaatat cgcgcagcgc tgaaaacggc ggcagccgct ctggaagagt accagggcgt
     2521 gaccacccgc cagctgagcg aagtcctgcg tcacggtctg cgtgagagct aagcttggcc
     2581 tatgcggccg ctaagggtcg acctgcagcc aagcttaatt agctgagctt ggactcctgt
     2641 tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc
     2701 gccgggcgtt ttttattggt gagaatccaa gctaggggaa ttcatcgtga ctgactgacg
     2761 atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     2821 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     2881 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     2941 tataattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     3001 ataataatag tttcttagaa tcctgatgtc cggcggtgct tttgccgtta cgcaccaccc
     3061 cgtcagtagc tgaacaggag ggacagctga tagaaacaga agccactgga gcacctcaaa
     3121 aacaccatca tacactaaat cagtaagttg gcagcatcac ccgacgcact ttgcgccgaa
     3181 taaatacctg tgacggaaga tcacttcgca gaataaataa atcctggtgt ccctgttgat
     3241 accgggaagc cctgggccaa cttttggcga aaatgagacg ttgatcggca cgtaagaggt
     3301 tccaactttc accataatga aataagatca ctaccgggcg tattttttga gttatcgaga
     3361 ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat
     3421 atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc
     3481 tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag
     3541 cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa
     3601 ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttat
     3661 accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat
     3721 ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga gaacctggcc
     3781 tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt
     3841 ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc
     3901 atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat
     3961 catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc
     4021 gatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc ttaaacgcct
     4081 ggttgctacg cctgaataag tgataataag cggatgaatg gcagaaattc gaaagcaaat
     4141 tcgacccggt cgtcggttca gggcagggtc gttaaatagc cgcttatgtc tattgctggt
     4201 ttaccggttt attgactacc ggaagcagtg tgaccgtgtg cttctcaaat gcctgaggcc
     4261 agtttgctca ggctctcccc gtggaggtaa taattgacga tatgatcatt tattctgcct
     4321 cccagagcct gataaaaacg gttaccggat cgagtcaggc aactatggat gaacgaaata
     4381 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4441 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4501 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4561 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4621 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4681 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4741 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4801 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4861 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4921 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4981 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5041 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5101 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5161 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5221 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5281 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5341 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     5401 gcggtatttc acaccgcata aattccgaca ccatcgaatg gcgcaaaacc tttcgcggta
     5461 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     5521 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     5581 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     5641 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     5701 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     5761 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     5821 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     5881 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     5941 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     6001 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     6061 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     6121 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     6181 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     6241 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     6301 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     6361 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     6421 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     6481 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     6541 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     6601 aatgtaagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     6661 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     6721 tacggattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     6781 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     6841 caccgatcgc ccttcccaac agttgc
//
