LOCUS       Exported                7006 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  IPTG-inducible expression of p50-ligase fusion for protein 
            purification.
ACCESSION   .
VERSION     .
KEYWORDS    pCA24N-p50-ligase
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7006)
  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 7006)
  AUTHORS   .
  TITLE     Direct Submission
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"
FEATURES             Location/Qualifiers
     source          1..7006
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        80..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      2772..2866
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2936..2958)
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     promoter        3422..3524
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3525..4184
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3591..3610)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      4735..5323
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5224..5243
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5477..5494
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5567..5644
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5645..6727
                     /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(5664..5683)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        6776..6806
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6814..6830
                     /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     6819..6841
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6838..6854
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6838..6854
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6870..6887)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6870..6886)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6879..6901)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"