LOCUS       sequence-174184-        6910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG-inducible expression of ligase-NLS-cTF fusion for protein 
            purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6910)
  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 6910)
  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-NLS-cTF
FEATURES             Location/Qualifiers
     source          1..6910
                     /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      2676..2770
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2840..2862)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3326..3428
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3429..4088
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3495..3514)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      4639..5227
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5128..5147
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5381..5398
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5471..5548
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5549..6631
                     /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(5568..5587)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6680..6685,6686..6703,6704..6710)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6718..6734
                     /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     6742..6758
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6742..6758
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6774..6791)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6774..6790)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6783..6805)
                     /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 ctaggcaccc cgaccccgac cccgaccccg accggcgaat ttgactggca
     1741 gctcccgtca cattctggtc catacgagct tcggatcgag gtgcagccca agtctcacca
     1801 cagggctcac tatgagacgg aaggcagccg gggggctgtg aaggcttcag ctggaggaca
     1861 ccccattgtg cagctacacg gttacttgga gaatgaacct ctcacgctac agctgttcat
     1921 tgggacggct gacgaccgcc tgctgaggcc ccacgccttc taccaggtcc accggatcac
     1981 ggggaagact gtctccacca ccagccacga gatcatcctg tccaacacca aagtcctgga
     2041 gatcccgttg cttccagaaa ataacatgcg agccatcatc gactgtgctg ggatcctgaa
     2101 gctcagaaac tctgatattg agctgaggaa aggggagaca gacatcggga ggaagaacac
     2161 cagggtgagg ctggtcttcc gagttcacat cccacagccc aatggccgga cgctgtctct
     2221 ccaggtggcc tcgaacccta tcgagtgttc ccagcggtca gcatccaact tgaaaattgt
     2281 aagaatggac aggacagctg gatgtgtgac tggaggggag gaaatttatc ttctttgtga
     2341 caaagttcag aaagatgaca tccagattcg attttatgaa gaggaagaaa atggtggagt
     2401 ctgggaagga tttggagatt tttcccccac agatgttcat agacaatttg ccattgtctt
     2461 caaaactcca aagtataaag atattaatat tacaaaacca gcctctgtgt ttgtccagct
     2521 tcggaggaaa tctgacttgg aaaccagtga accaaaacct ttcctctact atcctgaaat
     2581 caaagataaa gaagaagtgc agaggaaacg tcagaagtaa ggcctatgcg gccgctaagg
     2641 gtcgacctgc agccaagctt aattagctga gcttggactc ctgttgatag atccagtaat
     2701 gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg cgttttttat
     2761 tggtgagaat ccaagctagg ggaattcatc gtgactgact gacgatctgc ctcgcgcgtt
     2821 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     2881 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     2941 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtataat tcttgaagac
     3001 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atagtttctt
     3061 agaatcctga tgtccggcgg tgcttttgcc gttacgcacc accccgtcag tagctgaaca
     3121 ggagggacag ctgatagaaa cagaagccac tggagcacct caaaaacacc atcatacact
     3181 aaatcagtaa gttggcagca tcacccgacg cactttgcgc cgaataaata cctgtgacgg
     3241 aagatcactt cgcagaataa ataaatcctg gtgtccctgt tgataccggg aagccctggg
     3301 ccaacttttg gcgaaaatga gacgttgatc ggcacgtaag aggttccaac tttcaccata
     3361 atgaaataag atcactaccg ggcgtatttt ttgagttatc gagattttca ggagctaagg
     3421 aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc
     3481 gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc
     3541 agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg
     3601 cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga
     3661 aagacggtga gctggtgata tgggatagtg ttcacccttg ttataccgtt ttccatgagc
     3721 aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac
     3781 acatatattc gcaagatgtg gcgtgttacg gtgagaacct ggcctatttc cctaaagggt
     3841 ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt
     3901 taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata
     3961 cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg
     4021 gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg
     4081 gggcgtaatt tttttaaggc agttattggt gcccttaaac gcctggttgc tacgcctgaa
     4141 taagtgataa taagcggatg aatggcagaa attcgaaagc aaattcgacc cggtcgtcgg
     4201 ttcagggcag ggtcgttaaa tagccgctta tgtctattgc tggtttaccg gtttattgac
     4261 taccggaagc agtgtgaccg tgtgcttctc aaatgcctga ggccagtttg ctcaggctct
     4321 ccccgtggag gtaataattg acgatatgat catttattct gcctcccaga gcctgataaa
     4381 aacggttacc ggatcgagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     4441 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     4501 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4561 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4621 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4681 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4741 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     4801 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4861 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4921 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4981 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5041 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5101 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5161 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5221 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5281 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5341 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     5401 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     5461 cataaattcc gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc
     5521 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     5581 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     5641 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     5701 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     5761 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     5821 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     5881 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     5941 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     6001 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     6061 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     6121 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     6181 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     6241 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     6301 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     6361 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     6421 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     6481 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     6541 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     6601 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     6661 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     6721 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgga ttcactggcc
     6781 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     6841 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     6901 caacagttgc
//
