LOCUS       sequence-174175-        6901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG-inducible expression of ligase-cTF fusion for protein 
            purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6901)
  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 6901)
  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-cTF
FEATURES             Location/Qualifiers
     source          1..6901
                     /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      2667..2761
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2831..2853)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3317..3419
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3420..4079
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3486..3505)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      4630..5218
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5119..5138
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5372..5389
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5462..5539
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5540..6622
                     /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(5559..5578)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6671..6676,6677..6694,6695..6701)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6709..6725
                     /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     6733..6749
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6733..6749
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6765..6782)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6765..6781)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6774..6796)
                     /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 ggcgactggc agctcccgtc
     1741 acattctggt ccatacgagc ttcggatcga ggtgcagccc aagtctcacc acagggctca
     1801 ctatgagacg gaaggcagcc ggggggctgt gaaggcttca gctggaggac accccattgt
     1861 gcagctacac ggttacttgg agaatgaacc tctcacgcta cagctgttca ttgggacggc
     1921 tgacgaccgc ctgctgaggc cccacgcctt ctaccaggtc caccggatca cggggaagac
     1981 tgtctccacc accagccacg agatcatcct gtccaacacc aaagtcctgg agatcccgtt
     2041 gcttccagaa aataacatgc gagccatcat cgactgtgct gggatcctga agctcagaaa
     2101 ctctgatatt gagctgagga aaggggagac agacatcggg aggaagaaca ccagggtgag
     2161 gctggtcttc cgagttcaca tcccacagcc caatggccgg acgctgtctc tccaggtggc
     2221 ctcgaaccct atcgagtgtt cccagcggtc agcatccaac ttgaaaattg taagaatgga
     2281 caggacagct ggatgtgtga ctggagggga ggaaatttat cttctttgtg acaaagttca
     2341 gaaagatgac atccagattc gattttatga agaggaagaa aatggtggag tctgggaagg
     2401 atttggagat ttttccccca cagatgttca tagacaattt gccattgtct tcaaaactcc
     2461 aaagtataaa gatattaata ttacaaaacc agcctctgtg tttgtccagc ttcggaggaa
     2521 atctgacttg gaaaccagtg aaccaaaacc tttcctctac tatcctgaaa tcaaagataa
     2581 agaagaagtg cagaggaaac gtcagaagta aggcctatgc ggccgctaag ggtcgacctg
     2641 cagccaagct taattagctg agcttggact cctgttgata gatccagtaa tgacctcaga
     2701 actccatctg gatttgttca gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa
     2761 tccaagctag gggaattcat cgtgactgac tgacgatctg cctcgcgcgt ttcggtgatg
     2821 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     2881 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     2941 cagccatgac ccagtcacgt agcgatagcg gagtgtataa ttcttgaaga cgaaagggcc
     3001 tcgtgatacg cctattttta taggttaatg tcatgataat aatagtttct tagaatcctg
     3061 atgtccggcg gtgcttttgc cgttacgcac caccccgtca gtagctgaac aggagggaca
     3121 gctgatagaa acagaagcca ctggagcacc tcaaaaacac catcatacac taaatcagta
     3181 agttggcagc atcacccgac gcactttgcg ccgaataaat acctgtgacg gaagatcact
     3241 tcgcagaata aataaatcct ggtgtccctg ttgataccgg gaagccctgg gccaactttt
     3301 ggcgaaaatg agacgttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa
     3361 gatcactacc gggcgtattt tttgagttat cgagattttc aggagctaag gaagctaaaa
     3421 tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac
     3481 attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata
     3541 ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc
     3601 acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg
     3661 agctggtgat atgggatagt gttcaccctt gttataccgt tttccatgag caaactgaaa
     3721 cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt
     3781 cgcaagatgt ggcgtgttac ggtgagaacc tggcctattt ccctaaaggg tttattgaga
     3841 atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg
     3901 ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg
     3961 acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg
     4021 tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaat
     4081 ttttttaagg cagttattgg tgcccttaaa cgcctggttg ctacgcctga ataagtgata
     4141 ataagcggat gaatggcaga aattcgaaag caaattcgac ccggtcgtcg gttcagggca
     4201 gggtcgttaa atagccgctt atgtctattg ctggtttacc ggtttattga ctaccggaag
     4261 cagtgtgacc gtgtgcttct caaatgcctg aggccagttt gctcaggctc tccccgtgga
     4321 ggtaataatt gacgatatga tcatttattc tgcctcccag agcctgataa aaacggttac
     4381 cggatcgagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     4441 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4501 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4561 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4621 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4681 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4741 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     4801 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4861 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4921 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4981 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5041 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5101 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5161 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5221 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     5281 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     5341 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     5401 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcataaattc
     5461 cgacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     5521 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     5581 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     5641 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     5701 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     5761 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     5821 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     5881 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     5941 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     6001 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     6061 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     6121 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     6181 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     6241 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     6301 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
     6361 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     6421 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     6481 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     6541 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     6601 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     6661 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     6721 aacaatttca cacaggaaac agctatgacc atgattacgg attcactggc cgtcgtttta
     6781 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     6841 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     6901 c
//
