LOCUS       62056_12140        7889 bp DNA     circular SYN 14-DEC-2020
DEFINITION  Cloning vector pHal2-FAPG462VRFP, complete sequence.
ACCESSION   MW076828
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7889)
  AUTHORS   Trisrivirat D, Hughes JMX., Hoeven R, Faulkner M, Toogood H, Chaiyen
            P, Scrutton NS.
  TITLE     Promoter engineering for microbial bio-alkane gas production
  JOURNAL   Synth Biol (Oxf) 5 (1), ysaa022 (2020)
  PUBMED    33263086
REFERENCE   2  (bases 1 to 7889)
  AUTHORS   Trisrivirat D, Hughes JMX., Hoeven R, Faulkner M, Toogood HS, 
            Chaiyen P, Scrutton NS.
  TITLE     Direct Submission
  JOURNAL   Submitted (06-OCT-2020) Chemistry, University of Manchester, 131 
            Princess Street, Manchester, Lancashire M1 7DN, United Kingdom
REFERENCE   3  (bases 1 to 7889)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Synth Biol 
            (Oxf)"; date: "2020"; volume: "5"; issue: "1"; pages: "ysaa022"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (06-OCT-2020) Chemistry, University of Manchester, 131 Princess 
            Street, Manchester, Lancashire M1 7DN, United Kingdom"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1028..1130
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     CDS             1350..2138
                     /codon_start=1
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     oriT            2319..2427
                     /label=oriT
                     /note="incP origin of transfer"
     rep_origin      2521..3109
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3166..3996)
                     /codon_start=1
                     /label=pRO1600 Rep
                     /note="replication protein for the broad-host-range plasmid
                     pRO1600 from Pseudomonas aeruginosa"
                     /translation="VASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     rep_origin      4010..4361
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     terminator      complement(4434..4520)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     protein_bind    complement(4637..4656)
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     misc_RNA        4658..4708
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             4758..4780
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6605..7279
                     /codon_start=1
                     /label=mRFP1
                     /note="monomeric derivative of DsRed (Campbell et al.,
                     2002)"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             7295..7339
                     /codon_start=1
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /translation="KETAAAKFERQHMDS"
     terminator      7448..7542
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             join(7651..7889,1..574)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
       61 gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
      121 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
      181 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
      241 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
      301 agaaatgcat aagcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
      361 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
      421 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
      481 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
      541 attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta
      601 acactggcag agcattacgc tgacttgacg ggacggcggc tttgttgaat aaatcgaact
      661 tttgctgagt tgaaggatca gatcacgcat cttcccgaca acgcagaccg ttccgtggca
      721 aagcaaaagt tcaaaatcac caactggtcc acctacaaca aagctctcat caaccgtggc
      781 tccctcactt tctggctgga tgatggggcg attcaggcct ggtatgagtc agcaacacct
      841 tcttcacgag gcagacctca gcgctattct gaccttgcca tcacgactgt gctggtcatt
      901 aaacgcgtat tcaggctgac cctgcgcgct gcgcagggct ttattgattc catttttaca
      961 ctgatgaatg ttccgttgcg ctgcccggat tacagccgga tcctctagag tcgacctgca
     1021 ggcatgctga tcggcacgta agaggttcca actttcacca taatgaaata agatcactac
     1081 cgggcgtatt ttttgagtta tcgagatttt caggagctaa ggaagctaaa atgcgctcac
     1141 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca
     1201 tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag
     1261 cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt acgcagcagg
     1321 gcagtcgccc taaaacaaag ttaaacatca tgagggaagc ggtgatcgcc gaagtatcga
     1381 ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg
     1441 tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc
     1501 tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt
     1561 tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg
     1621 ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag
     1681 aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc
     1741 tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg
     1801 aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa
     1861 cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt
     1921 cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact
     1981 gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct agacaggctt
     2041 atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc
     2101 actacgtgaa aggcgagatc accaaggtag tcggcaaata aactagtaaa taataaaaaa
     2161 gccggattaa taatctggct ttttatattc tctgcataac cctgcttcgg ggtcattata
     2221 gcgatttttt cggtatatcc atcctttttc gcacgatata caggattttg ccaaagggtt
     2281 cgtgtagact ttccttggtg tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc
     2341 ccacccgcga gcgggtgttc cttcttcact gtcccttatt cgcacctggc ggtgctcaac
     2401 gggaatcctg ctctgcgagg ctggccgtag gccggccgat aatctcatga ccaaaatccc
     2461 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2521 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2581 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2641 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     2701 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2761 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2821 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2881 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2941 gagaaaggcg gacaggcatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3001 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3061 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3121 cgcggccgtg aaaggcaggc cggtccgtgg tggccacggc ctctaggcca gatccagcgg
     3181 catctgggtt agtcgagcgc gggccgcttc ccatgtctca ccagggcgag cctgtttcgc
     3241 gatctcagca tctgaaatct tcccggcctt gcgcttcgct ggggccttac ccaccgcctt
     3301 ggcgggcttc ttcggtccaa aactgaacaa cagatgtgtg accttgcgcc cggtctttcg
     3361 ctgcgcccac tccacctgta gcgggctgtg ctcgttgatc tgcgtcacgg ctggatcaag
     3421 cactcgcaac ttgaagtcct tgatcgaggg ataccggcct tccagttgaa accactttcg
     3481 cagctggtca atttctattt cgcgctggcc gatgctgtcc cattgcatga gcagctcgta
     3541 aagcctgatc gcgtgggtgc tgtccatctt ggccacgtca gccaaggcgt atttggtgaa
     3601 ctgtttggtg agttccgtca ggtacggcag catgtctttg gtgaacctga gttctacacg
     3661 gccctcaccc tcccggtaga tgattgtttg cacccagccg gtaatcatca cactcggtct
     3721 tttccccttg ccattgggct cttgggttaa ccggacttcc cgccgtttca ggcgcagggc
     3781 cgcttctttg agctggttgt aggaagattc gatagggaca cccgccatcg tcgctatgtc
     3841 ctccgccgtc actgaataca tcacttcatc ggtgacaggc tcgctcctct tcacctggct
     3901 aatacaggcc agaacgatcc gctgttcctg aacactgagg cgatacgcgg cctcgaccag
     3961 ggcattgctt ttgtaaacca ttgggggtga ggccacgttc gacattcctt gtgtataagg
     4021 ggacactgta tctgcgtccc acaatacaac aaatccgtcc ctttacaaca acaaatccgt
     4081 cccttcttaa caacaaatcc gtcccttaat ggcaacaaat ccgtcccttt ttaaactcta
     4141 gaggccacgg attacgtggc ctgtagacgt cctaaaaggt ttaaaaggga aaaggaagaa
     4201 aagggtggaa acgcaaaaaa cgcaccacta cgtggccccg ttggggccgc atttgtgccc
     4261 ctgaaggggc gggggaggcg tctgggcaat ccccgtttta ccagtcccct atcgccgcct
     4321 gagagggcgc aggaagcgag taatcagggt atcgaggcgg attcaccctt ggcgtccaac
     4381 cagcggcacc agcggcgcct gagaggggcg cgcccagctg tctagggcgg cggatttgtc
     4441 ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac
     4501 tgagcctttc gttttatttg atgcctttaa ttaaagcgga taacaatttc acacaggagg
     4561 ccgcctaggc aaggatgcct ccacaccgct cgtcacatcc tgcccatgag ttaattatat
     4621 ttgtggcatt atagggaatt gtgagcgctc acaattagct gtcaccggat gtgctttccg
     4681 gtctgatgag tccgtgagga cgaaacagcc tctacaaata attttgttta atactagaga
     4741 aagaggagaa atactagttt gtttaacttt aagaaggaga tataccatgg ccagcgcagt
     4801 tgaagatatt cgtaaagttc tgagcgatag cagcagtccg gttgcaggtc agaaatatga
     4861 ttatattctg gttggtggtg gcaccgcagc atgtgttctg gcaaatcgtc tgagcgcaga
     4921 tggtagcaaa cgtgttctgg ttctggaagc aggtccggat aataccagcc gtgatgttaa
     4981 aattccggca gcaattaccc gtctgtttcg tagtccgctg gattggaacc tgtttagcga
     5041 actgcaagaa cagctggcag aacgtcagat ttatatggca cgtggtcgtc tgttaggtgg
     5101 tagcagcgca accaatgcaa ccctgtatca tcgtggtgca gccggtgatt atgatgcatg
     5161 gggtgttgaa ggttggagca gcgaagatgt tctgagctgg tttgttcagg cagaaaccaa
     5221 tgccgatttt ggtccgggtg catatcatgg tagcggtggt ccgatgcgtg ttgaaaatcc
     5281 gcgttatacc aataaacagc tgcataccgc atttttcaaa gcagcagaag aagttggtct
     5341 gaccccgaat agcgatttta atgattggag ccatgatcac gcaggttatg gcacctttca
     5401 ggttatgcag gataaaggca cccgtgcaga tatgtatcgt cagtatctga aaccggttct
     5461 gggtcgtcgt aatctgcagg ttctgaccgg tgcagcagtt accaaagtta atattgatca
     5521 ggcagcaggt aaagcacagg cactgggcgt tgaattttca accgatggtc cgaccggtga
     5581 acgtctgtca gcagaactgg cacctggtgg tgaagttatt atgtgtgccg gtgcagttca
     5641 taccccgttt ctgctgaaac atagcggtgt tggtccgagc gcagaactga aagaatttgg
     5701 tattccggtt gttagcaatc tggcaggcgt tggccagaat ctgcaagatc agcctgcatg
     5761 tctgaccgca gcaccggtta aagaaaaata tgatggtatt gccatcagcg accacatcta
     5821 taatgaaaaa ggtcagattc gcaaacgtgc cattgcaagc tatctgcttg gtggtcgtgg
     5881 tggcctgacc agtaccggtt gtgatcgcgg tgcatttgtt cgtaccgcag gtcaggcact
     5941 gccggatctg caagttcgtt ttgttcctgg tatggcactg gatccggatg ttgttagcac
     6001 ctatgtgcgt tttgccaaat ttcagagcca gggcctgaaa tggccgagcg gtattaccat
     6061 gcagctgatt gcatgtcgtc cgcagagtac cggtagcgtg ggtctgaaaa gcgcagatcc
     6121 gtttgcaccg cctaaactga gtccgggtta tctgaccgat aaagatggtg cagatctggc
     6181 gaccctgcgt aaaggtattc attgggcacg tgatgttgca cgtagctcag cactgagcga
     6241 atatctggat ggtgaactgt ttcctggtag tggtgttgtt agtgatgatc agatcgatga
     6301 atatatccgt cgtagcattc atagcagcaa tgcaattacc ggcacctgta aaatgggtaa
     6361 tgccggtgat agcagctcag ttgttgataa ccagctgcgt gtgcatggtg tggaaggtct
     6421 gcgtgttgtt gatgcaagcg ttgttccgaa aattcctggt ggtcagacag gtgctccggt
     6481 ggtgatgatt gcagaacgtg cagcagccct gctgaccggt aaagcaacca ttggtgcaag
     6541 cgcagccgca ccggcaaccg ttgcagcagg ttctgcgggt tctgcggccg gttctggcga
     6601 atttatggcg agtagcgaag acgttatcaa agagttcatg cgtttcaaag ttcgtatgga
     6661 aggttccgtt aacggtcacg agttcgaaat cgaaggtgaa ggtgaaggtc gtccgtacga
     6721 aggtacccag accgctaaac tgaaagttac caaaggtggt ccgctgccgt tcgcttggga
     6781 catcctgtcc ccgcagttcc agtacggttc caaagcttac gttaaacacc cggctgacat
     6841 cccggactac ctgaaactgt ccttcccgga aggtttcaaa tgggaacgtg ttatgaactt
     6901 cgaagacggt ggtgttgtta ccgttaccca ggactcctcc ctgcaagacg gtgagttcat
     6961 ctacaaagtt aaactgcgtg gtaccaactt cccgtccgac ggtccggtta tgcagaaaaa
     7021 aaccatgggt tgggaagctt ccaccgaacg tatgtacccg gaagacggtg ctctgaaagg
     7081 tgaaatcaaa atgcgtctga aactgaaaga cggtggtcac tacgacgctg aagttaaaac
     7141 cacctacatg gctaaaaaac cggttcagct gccgggtgct tacaaaaccg acatcaaact
     7201 ggacatcacc tcccacaacg aagactacac catcgttgaa cagtacgaac gtgctgaagg
     7261 tcgtcactcc accggtgctt aactcgagtc tggtaaagaa accgctgctg cgaaatttga
     7321 acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg ctgccaccgc
     7381 tgagcaataa agcccatgca agcttgcggc cgcgtcgtga ctgggaaaac cctggcgact
     7441 agtcttggac tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc
     7501 agaacgctcg gttgccgccg ggcgtttttt attggtgaga atccagacgt tgtgtctcaa
     7561 aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct
     7621 gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc
     7681 tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
     7741 gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca
     7801 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     7861 agactaaact ggctgacgga atttatgcc
//
