LOCUS       62056_6220        8800 bp DNA     circular SYN 18-AUG-2021
DEFINITION  Expression vector pCF, complete sequence.
ACCESSION   MZ567118
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8800)
  AUTHORS   Fang L, Fan J, Luo S, Chen Y, Wang C, Cao Y, Song H.
  TITLE     Genome-scale target identification in Escherichia coli for 
            high-titer production of free fatty acids
  JOURNAL   Nat Commun 12 (1), 4976 (2021)
REFERENCE   2  (bases 1 to 8800)
  AUTHORS   Fang L, Fan J, Luo S, Chen Y, Wang C, Cao Y, Song H.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-JUL-2021) School of Chemical Engineering, Tianjin 
            University, No. 135, Yaguan Road, Haihe Education Park, Jinnan 
            District, Tianjin, Tian Jin, Tian Jin 300350, China
REFERENCE   3  (bases 1 to 8800)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun"; date: "2021"; volume: "12"; issue: "1"; pages: "4976"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (13-JUL-2021) School of Chemical Engineering, Tianjin University, 
            No. 135, Yaguan Road, Haihe Education Park, Jinnan District, 
            Tianjin, Tian Jin, Tian Jin 300350, China"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..8800
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(120..776)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(777..879)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(1405..1949)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     protein_bind    complement(2140..2161)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(2177..3256)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(3257..3334)
                     /label=lacI promoter
     promoter        3405..3434
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    3442..3458
                     /label=lac operator
                     /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)."
     misc_feature    3475..3544
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region
                     (Berg et al., 1989)"
     CDS             3595..3618
                     /label=minicistron
                     /note="synthetic cistron containing a ribosome binding site
                     (Shine-Dalgarno sequence), for enhancing the bacterial 
                     expression of a downstream cistron (Schoner, 1997)"
     CDS             3625..7728
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     terminator      7780..7826
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     promoter        7898..7916
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7917..7941
                     /label=lac operator
                     /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)."
     gene            7984..8535
                     /gene="tesA'"
                     /label=tesA'
     CDS             7984..8535
                     /codon_start=1
                     /transl_table=11
                     /gene="tesA'"
                     /product="TesA'"
                     /label=tesA'
                     /note="truncated fatty acyl-ACP thioesterase TesA"
                     /protein_id="QXV67745.1"
                     /translation="MADTLLILGDSLSAGYRMSASAAWPALLNDKWQSKTSVVNASISG
                     DTSQQGLARLPALLKQHQPRWVLVELGGNDGLRGFQPQQTEQTLRQILQDVKAANAEPL
                     LMQIRLPANYGRRYNEAFSAIYPKLAKEFDVPLLPFFMEEVYLKPQWMQDDGIHPNRDA
                     QPFIADWMAKQLQPLVNHDS"
     CDS             8554..8598
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     terminator      8650..8697
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ctgaaccgac gaccgggtcg aatttgcttt cgaatttctg ccattcatcc gcttattatc
       61 acttattcag gcgtagcacc aggcgtttaa gggcaccaat aactgcctta aaaaaattac
      121 gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg
      181 aagccatcac agacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct
      241 tgcgtataat atttgcccat agtgaaaacg ggggcgaaga agttgtccat attggccacg
      301 tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca
      361 ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat
      421 atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca
      481 gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg
      541 tctttcattg ccatacggaa ctccggatga gcattcatca ggcgggcaag aatgtgaata
      601 aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc
      661 agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct
      721 ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta
      781 gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt
      841 tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccaaaag
      901 ttggcccagg gcttcccggt atcaacaggg acaccaggat ttatttattc tgcgaagtga
      961 tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc caacttactg
     1021 atttagtgta tgatggtgtt tttgaggtgc tccagtggct tctgtttcta tcagctgtcc
     1081 ctcctgttca gctactgacg gggtggtgcg taacggcaaa agcaccgccg gacatcagcg
     1141 ctagcggagt gtatactggc ttactatgtt ggcactgatg agggtgtcag tgaagtgctt
     1201 catgtggcag gagaaaaaag gctgcaccgg tgcgtcagca gaatatgtga tacaggatat
     1261 attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggaaa
     1321 tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt aacagggaag
     1381 tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca agcatcacga
     1441 aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     1501 tcccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt
     1561 ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt
     1621 tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc
     1681 cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc
     1741 cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg
     1801 aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg
     1861 tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag
     1921 agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt
     1981 tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat
     2041 ataagttgta attctcatgt tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     2101 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     2161 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     2221 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     2281 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     2341 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     2401 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg
     2461 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     2521 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     2581 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     2641 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     2701 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     2761 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     2821 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     2881 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     2941 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     3001 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     3061 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     3121 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     3181 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     3241 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     3301 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     3361 tgcagacatc ataacggttc tggcaaatat tctgaaatga gctgttgaca attaatcatc
     3421 cggctcgtat aatgtgtgga attgtgagcg gataacaatt tcacacagga aacagcgccg
     3481 ctgagaaaaa gcgaagcggc actgctcttt aacaatttat cagacaatct gtgtgggcac
     3541 tcgaccggaa ttatcgatta actttattat taaaaattaa agaggtatat attaatgtat
     3601 cgattaaata aggaggaata aaccatggac aaaaaatact ctatcggtct ggctatcggt
     3661 actaactctg ttggttgggc tgttatcacc gacgaataca aagttccgtc taaaaaattc
     3721 aaagttctgg gtaacaccga ccgtcactct atcaaaaaaa acctgatcgg tgctctgctg
     3781 ttcgactctg gtgaaaccgc tgaagctacc cgtctgaaac gtaccgctcg tcgtcgttac
     3841 acccgtcgta aaaaccgtat ctgctacctg caagaaatct tctctaacga aatggctaaa
     3901 gttgacgact ctttcttcca ccgtctcgaa gaatcgttcc tggttgaaga ggacaaaaaa
     3961 cacgaacgtc acccgatctt cggtaacatc gttgacgaag ttgcttacca cgaaaaatac
     4021 ccgaccatct accacctgcg taaaaaactg gttgactcta ccgacaaagc tgacctgcgt
     4081 ctgatctacc tggctctggc tcacatgatc aaattccgtg gtcacttcct gatcgaaggt
     4141 gacctgaacc cggacaactc tgacgttgac aaactgttca tccagctggt tcagacctac
     4201 aaccagctgt tcgaagaaaa cccgatcaac gcttctggtg ttgacgctaa agctatcctg
     4261 tctgctcgtc tgtctaaatc tcgtcgtctg gaaaacctga tcgctcagct gccgggtgaa
     4321 aaaaaaaacg gtctgttcgg taacctgatc gctctgtctc tgggtctgac cccgaacttc
     4381 aaatctaact tcgacctggc tgaagacgct aaactgcaac tgtctaaaga cacctacgac
     4441 gacgacctgg acaacctgct ggctcagatc ggtgaccagt acgctgacct gttcctggct
     4501 gctaaaaacc tgtctgacgc tatcctgctg tctgacatcc tgcgtgttaa caccgaaatc
     4561 accaaagctc cgctgtctgc ttctatgatc aaacgttacg acgaacacca ccaggacctg
     4621 accctgctga aagctctggt tcgtcagcag ctgccggaaa aatacaaaga aatcttcttc
     4681 gaccagtcta aaaacggtta cgctggttac atcgacggtg gtgcttctca ggaagaattt
     4741 tacaaattca tcaaaccgat cctggaaaaa atggacggta ctgaagaact gctggttaaa
     4801 ctgaaccgtg aagacctgct gcgtaaacag cgtaccttcg acaacggttc tatcccgcac
     4861 cagatccacc tgggtgaact gcacgctatc ctgcgtcgtc aggaagactt ctacccgttc
     4921 ctgaaagaca accgtgaaaa aatcgaaaaa atcctgacct tccgtatccc gtactacgtt
     4981 ggtccgctgg ctcgtggtaa ctctcgtttc gcttggatga cccgtaaatc tgaagaaacc
     5041 atcaccccgt ggaacttcga agaagttgtt gacaaaggtg cttctgctca gtctttcatc
     5101 gaacgtatga ccaacttcga caaaaacctg ccgaacgaaa aagttctgcc gaaacactct
     5161 ctgctgtacg aatacttcac cgtttacaac gaactgacca aagttaaata cgttaccgaa
     5221 ggtatgcgta aaccggcttt cctgtctggt gaacagaaaa aagctatcgt tgacctgctg
     5281 ttcaaaacca accgtaaagt taccgttaaa cagctgaaag aagactactt caaaaaaatc
     5341 gaatgcttcg actctgttga aatctctggt gttgaagacc gtttcaacgc ttctctgggt
     5401 acttaccacg acctgctgaa aatcatcaaa gacaaagact tcctggacaa cgaagaaaac
     5461 gaagacatcc tggaagacat cgttctgacc ctgaccctgt tcgaagaccg tgaaatgatc
     5521 gaagaacgtc tgaaaaccta cgctcacctg ttcgacgaca aagttatgaa acagctgaaa
     5581 cgtcgtcgtt acaccggttg gggtcgtctg tctcgtaaac tgatcaacgg tatccgtgac
     5641 aaacagtctg gtaaaaccat cctggacttc ctgaaatctg acggtttcgc taaccgtaac
     5701 ttcatgcagc tgatccacga cgactctctg accttcaaag aagacatcca gaaggcgcag
     5761 gtaagcggtc agggtgactc tctgcacgaa cacatcgcta acctggctgg ttctccggct
     5821 atcaaaaaag gtatcctgca aaccgttaaa gttgttgacg aactggttaa agttatgggt
     5881 cgtcacaaac cggaaaacat cgttatcgaa atggctcgtg aaaaccagac cacccagaaa
     5941 ggtcagaaaa actctcgtga acgtatgaaa cgtatcgaag aaggtatcaa agaactgggt
     6001 tctcagatcc tgaaagaaca cccggttgaa aacacccagc tgcaaaacga aaaactgtac
     6061 ctgtactacc tgcaaaacgg tcgtgacatg tacgttgacc aggaactgga catcaaccgt
     6121 ctgtctgact acgacgttga cgctatcgtt ccgcagtctt tcctgaaaga cgactctatc
     6181 gacaacaaag ttctgacccg ttctgacaaa aaccgtggta aatctgacaa cgttccgtct
     6241 gaagaagttg ttaaaaaaat gaaaaactac tggcgtcagc tgctgaacgc taaactgatc
     6301 acccagcgta aattcgacaa cctgaccaaa gctgaacgtg gtggtctgtc tgaactggac
     6361 aaagctggtt tcatcaaacg tcagctggtt gaaacccgtc agatcaccaa acacgttgct
     6421 cagatcctgg actctcgtat gaacaccaaa tacgacgaaa acgacaaact gatccgtgaa
     6481 gttaaagtta tcaccctgaa atctaaactg gtttctgact tccgtaaaga cttccagttc
     6541 tacaaagttc gtgaaatcaa caactaccac cacgctcacg acgcttacct gaacgctgtt
     6601 gttggtactg ctctgatcaa aaaatacccg aaactggagt cggaatttgt gtacggggac
     6661 tacaaagttt acgacgttcg taaaatgatc gctaaatctg aacaggaaat cggtaaagct
     6721 accgctaaat acttcttcta ctctaacatc atgaacttct tcaaaaccga aatcaccctg
     6781 gctaacggtg aaatccgtaa acgtccgctg atcgaaacca acggtgaaac cggcgagatc
     6841 gtctgggaca aaggccgtga cttcgctacc gttcgtaaag ttctgtctat gccgcaggtt
     6901 aacatcgtta aaaaaaccga agttcagacc ggtggtttct ctaaagaatc tatcctgccg
     6961 aaacgtaact ctgacaaact gatcgctcgt aaaaaagact gggacccaaa aaaatacggt
     7021 ggcttcgact ctccgactgt tgcttactct gttctggttg ttgctaaagt tgaaaaaggt
     7081 aaatctaaaa aactgaaatc tgttaaagaa ctgctgggta tcaccatcat ggaacgttct
     7141 tctttcgaaa aaaacccgat cgacttcctg gaagctaaag gttacaaaga agttaaaaaa
     7201 gacctgatca tcaaactgcc gaaatactct ctgttcgaac tggaaaacgg tcgtaaacgt
     7261 atgctggctt ctgctggtga actgcaaaaa ggtaacgaac tggctctgcc gtctaaatac
     7321 gttaacttcc tgtacctggc ttctcactac gaaaaactga aaggttctcc ggaagacaac
     7381 gaacagaaac agctgttcgt tgaacagcac aaacactacc tggacgaaat catcgaacag
     7441 atcagtgaat tttctaaacg tgttatcctg gctgacgcta acctggacaa agttctgtct
     7501 gcttacaaca aacaccgtga caaaccgatc cgtgaacagg ctgaaaacat catccacctg
     7561 ttcaccctga ccaacctcgg tgctccagct gcgttcaaat acttcgacac caccatcgac
     7621 cgtaaacgtt acacctctac caaagaagtt ctggacgcta ccctgatcca ccagtctatc
     7681 accggtctgt acgaaacccg tatcgacctg tctcagctgg gtggtgacta aggatccgag
     7741 ctccaattcg ccctatagtg agacgcgtgc tagaggcatc aaataaaacg aaaggctcag
     7801 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctta agtcgaacag
     7861 aaagtaatcg tattgtacac ggccgcataa tcgaaattaa tacgactcac tataggggaa
     7921 ttgtgagcgg ataacaattc cccatcttag tatattagtt aagtataaga aggagatata
     7981 catatggcgg acacgttatt gattctgggt gatagcctga gcgccgggta tcgaatgtct
     8041 gccagcgcgg cctggcctgc cttgttgaat gataagtggc agagtaaaac gtcggtagtt
     8101 aatgccagca tcagcggcga cacctcgcaa caaggactgg cgcgccttcc ggctctgctg
     8161 aaacagcatc agccgcgttg ggtgctggtt gaactgggcg gcaatgacgg tttgcgtggt
     8221 tttcagccac agcaaaccga gcaaacgctg cgccagattt tgcaggatgt caaagccgcc
     8281 aacgctgaac cattgttaat gcaaatacgt ctgcctgcaa actatggtcg ccgttataat
     8341 gaagccttta gcgccattta ccccaaactc gccaaagagt ttgatgttcc gctgctgccc
     8401 ttttttatgg aagaggtcta cctcaagcca caatggatgc aggatgacgg tattcatccc
     8461 aaccgcgacg cccagccgtt tattgccgac tggatggcga agcagttgca gcctttagta
     8521 aatcatgact cataaggtac cctcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa
     8581 cgccagcaca tggactcgtc tactagcgca gcttaattaa cctaggctgc tgccaccgct
     8641 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     8701 aaacctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta
     8761 aaccagcaat agacataagc ggctatttaa cgaccctgcc
//
