LOCUS       40924_4324        8325 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pAL23, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8325)
  AUTHORS   Karim AA, Gestaut DR, Fincker M, Ruth JC, Holmes EC, Sheu W, 
            Spormann AM.
  TITLE     Fine-tuned Protein Production in Methanosarcina acetivorans C2A
  JOURNAL   ACS Synth Biol (2018) In press
  PUBMED    29920209
REFERENCE   2  (bases 1 to 8325)
  AUTHORS   Karim AA.
  TITLE     Direct Submission
  JOURNAL   Submitted (30-NOV-2017) Civil and Environmental Engineering, 
            Stanford University, Y2E2, 473 Via Ortega, Stanford, CA 94305, USA
REFERENCE   3  (bases 1 to 8325)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8325)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol (2018) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (30-NOV-2017) Civil and Environmental Engineering, Stanford 
            University, Y2E2, 473 Via Ortega, Stanford, CA 94305, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8325
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..42
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    99..5569
                     /label=pC2A sequence
                     /note="pC2A sequence"
     CDS             complement(705..1649)
                     /codon_start=1
                     /gene="ssrA"
                     /product="site-specific recombinase A"
                     /label=ssrA
                     /protein_id="AWW92029.1"
                     /translation="MGEMAAIQLTGIYEPLDNERLISLFTSDCIGRGYSKRTIEGYVSH
                     AKFFLQMCGVNAGFPELEGFLVHIRDERQYTLATCNNYFASLSTFFDFLEFKHIIERNN
                     IPTFRKRYLRSYKKYHTPETRQLITIEDMAKLVKVPRYPMYQALIIFLAKTGIRRNELI
                     TLDRADIDLEKCTVRLKKTAKRSNRIVFFDLETKAFLEAYLNTRHDKEKALFIGRQNGH
                     RVQRNLVYDIVTSAASSIGLHNPEGRLEEHFTPHCCRHWFTTWLRRSGMERTFIQELRG
                     DSRGEAIDVYDHIEKDELREAYLKCIPQLGIKP"
     gene            complement(705..1649)
                     /gene="ssrA"
                     /label=ssrA
     misc_feature    2132..2779
                     /label=Orf2
                     /note="Orf2"
     misc_feature    complement(2785..3213)
                     /label=Orf1
                     /note="Orf1"
     CDS             complement(3292..4902)
                     /codon_start=1
                     /gene="repA"
                     /product="regulatory protein RepA"
                     /label=repA
                     /protein_id="AWW92030.1"
                     /translation="MSSDFSSRPGAVSYAPECRDFATCICTDGVLCPVRNKKNGVSICK
                     YYSCPDFDFSKCSKYRPLKVGDFVLPAKGKRPAYCGRPFTYAVSENYNARKDLNSHCNT
                     LSCPGCGILSELQHRFALVVILWTYALFSGQFPSWGFASMDFSRGVSVEAIRLFRRNLK
                     DRLERWGVTAGTTIFHPYRIKPEITRAIRKLTGLKGNDDSEAFWNYIRDDHNEGNLNKI
                     ADLLNIEINSIYDCLILAPHFHFFIFPGNIRITGKNPKSKKRCKSDEYDIFIQRLSHKV
                     NGKRQWELRSLMDIYKQVTYLYSHVGQLTDNRYGNIHIESRFGALFRWHPDKVVSSSGK
                     LTFEQLIDTRTEVLNLINEGREVKLVYNGELKWEGQENDDTGWIPLTKLRDHFSGGRQA
                     LHAYLKEAKAVKPEYADYLQQLIARNNFYHNLKGRDGKNKVPKKWRKLWASPFKVDELP
                     EFLKLEPAGSLLRVWFLEGLPDPPAGFLVVDSHQWEHDGCIIEESPEMPSNSLQKASEI
                     NYEELFTSLKSRSMMFGGD"
     gene            complement(3292..4902)
                     /gene="repA"
                     /label=repA
     regulatory      5566..5927
                     /label=mcr promoter from Methanococcus voltae
                     /note="mcr promoter from Methanococcus voltae"
                     /regulatory_class="promoter"
     CDS             5928..6524
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
     regulatory      6528..6867
                     /label=mcr terminator from Methanococcus voltae
                     /note="mcr terminator from Methanococcus voltae"
                     /regulatory_class="terminator"
     rep_origin      complement(6885..7273)
                     /direction=LEFT
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     regulatory      complement(7302..7334)
                     /label=bla terminator
                     /note="bla terminator"
                     /regulatory_class="terminator"
     CDS             complement(7338..8195)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8196..8300)
                     /label=AmpR promoter
ORIGIN
        1 ggatccgggc cctgtacaag atcctgtaaa acgacggcca gtgaattccc cgggaagctt
       61 cgccagggtt ttcccagtcg agctcgatat cggtacctct agtaagttat agaaatgtca
      121 tccgataccg aaaaagaaaa cggaataaat cagtaacaaa tcaaaaaaac aaaactataa
      181 catttaatct tcgtaaaatc aatatcaatc gaccatacac aaaacatgtt caaataaaat
      241 acatgggtgt acatatgtca accgacattg aaaaagaaaa cggaataaat cagtaacaaa
      301 tcaaaaaaac aaaaccataa catttctata acattccata gagtaaaaat caacatcgaa
      361 taaccttaca caaaccatac ataaaccata cataaaaata cacatatgtc aaccgttacc
      421 gtaaaaagat tacttaaata attttatact cacctcttat tttttgtgtt ttttattatc
      481 cagggcaacc tttcccaggg agaaattctt ccttgcagtt gcaggcaaga accaggactt
      541 tcttccaggg catactttcc cagggagaaa ttcttccttg cagctgcagg caagaaccag
      601 gactttcccc caggaccagg acctcaaggt ctgcatccag aggaagacct tcccagggcg
      661 ccaagatccc gcagattatg gaacattatt tcccgggaaa taattcaggg cttaatgccc
      721 aactgaggaa tacattttaa ataagcttct cttagttcat ccttttctat gtggtcgtac
      781 acgtctattg cttcgcctct cgaatctccc cttaattcct gaatgaatgt cctttccata
      841 cctgaacgtc gaagccatgt agtgaaccaa tgccggcagc aatgcggagt aaaatgttct
      901 tcaagtctcc cctcggggtt atggagtccg atagaggaag cggcagatgt gacaatatcg
      961 tacactaaat ttctttgtac acggtgaccg ttttgtctgc ctatgaataa tgctttttct
     1021 ttatcatgcc tcgtgttaag gtacgcttcc aggaaggcct tagtctcaag gtcaaagaat
     1081 acgatacgat ttgacctttt agctgtcttc ttgagtctca ccgtacactt ttccagatct
     1141 atgtcagctc gatcaagagt aattaattca tttctcctga tgccagtttt agctaaaaag
     1201 attatcaatg cttgatacat ggggtaacgt ggtactttga caagctttgc catgtcttca
     1261 atggtaatca attgccttgt ttccggagta tggtattttt tgtagcttct tagatagcgt
     1321 tttctgaacg tgggaatgtt gtttctctcg attatatgct tgaattcaag gaagtcaaaa
     1381 aaggtactca gtgaagcaaa gtaattgtta caggttgcga gtgtgtactg cctttcatct
     1441 cttatgtgaa caaggaaccc ttcaagctcc ggaaaccctg cattcactcc acacatttga
     1501 aggaaaaact tagcatgact tacgtatcct tctattgttc ttttagagta accccttccg
     1561 atacaatcag aagtgaataa acttataagt ctttcattat caagcggttc atagatacca
     1621 gttagctgaa tagctgccat ttcacccata aatcaataaa agaagtatgt aaataaatac
     1681 tttgtgcata gatgtcattt aaacacaaag tataagtaca tctaccttct tttagtatat
     1741 gctggtaaca gcaaaaaagc gccaaacaac gcatctttac ccaatagaat gacgtcgatt
     1801 aaaagagtaa gttggtggtg agccttaaaa aaggtaaaag agagcttact cttaagttaa
     1861 aaacggaatt ggcaaacagt aaggattgtt ccagcagtcc ttactaaaaa cattacttta
     1921 ttaattctat atagtcacaa cattcacaaa aataggcttt tttctcgatt tttccttttg
     1981 aatgtttata aagaaaaatc atatagcatt tattacactg tggacatatt ttcttttcaa
     2041 ttttgcccct tccaatttgt ggaagccttt tttcagtaac acgcttaggc ataatgaaag
     2101 atcggtgtat taagtattaa cattttctaa cttgtgcaat aataattatt cggggttgtc
     2161 gaagaacttc caaacagtag accgatccat tttccacatt attctaatga agccaagact
     2221 caaggatggg gggaaatatc cccccacacc ccccaatgag actacccaag acagcccgta
     2281 tgccgacagc gacacagtta aatatcaatg tgtattaata catatcaaca agtatcaaca
     2341 cacacaatta aacaataata cacatcaatg gagaacaata cacatgccaa cacaagaata
     2401 cacacggaca aacattacat taccagaaca tgttagagca atgattaaag agtacaataa
     2461 aagaagtaca tggggagacc tcaacgtatc agcagtatgc tctaatgcaa tagcagacag
     2521 aattagagct acattcccaa aattagaaaa agaactattt ccagaacaat ttccagaact
     2581 taacccaaaa atgacaaaaa ccagctcacc aggacaacca gaaataacac aaaaaggaaa
     2641 atccaaacaa ggagactcta aagatagaat atgcgcttat tgtggccaac cttttgaacc
     2701 gaaatcgcat aatcaaaaat tctgtaaaga tgcatgtaag tcagcaaatt atagaaaaaa
     2761 caaaaaaaca cagagttaaa cccattaacc acactctttt tttgaattta tctgcatttc
     2821 acggtacttt ttatccaaga aagcccacaa atcacgctct ttaaacggtt tcatggaaaa
     2881 attttcccgg aatgcattaa ttaacatctt ttcaatttta gggttgaaat aagccccata
     2941 acggtaatct ggcgaccaat ctgaagcttt aacctcttca taagtccgat acggcacatt
     3001 acccggaaga agcccaggaa ccacaagcag ataatagata tagcacctta tacacatatg
     3061 tgatttttta gtagtcttta accattcaat atatgaaaga tccgcacctg ttcgataatc
     3121 ctcaaaaaga attaagttcc tttgcctccg agatccacac acacagcaat acttcaattc
     3181 cataatacat aacaccccga aataataaga catctgaaaa accgtatcac tttaggcttt
     3241 aagcatctta tccggacctt cgagcccaaa agccttgttg aacgtggcat ctcaatcacc
     3301 cccaaacatc atggagcgtg attttaagct tgtaaatagc tcttcataat taatttcgga
     3361 agccttttgc aagctgttag aaggcatttc aggcgattct tcaattatac agccatcatg
     3421 ctcccattga tgagagtcaa ccaccaggaa gccggcaggg ggatcaggaa gcccttctaa
     3481 gaaccatacc cgaagaagac tcccggcagg ttcaagcttc aaaaactccg gtaattcatc
     3541 cactttgaag ggagaagccc ataatttacg ccatttttta ggaactttgt ttttgccatc
     3601 tcgccctttt aggttatgat aaaaattatt tcttgcaatt aattgttgca agtaatcggc
     3661 atattcaggt ttaaccgctt tagcttcctt caaataagca tgtagagcct gtcttccacc
     3721 cgaaaaatga tcccgaagct ttgttaacgg tatccatcca gtatcatcgt tttcctgtcc
     3781 ttcccacttc aactctccat tataaacgag ttttacttct cgaccttcat tgattaaatt
     3841 aaggacttcg gttctggtat ctatcaattg ctcaaatgtc agctttccgg aggaacttac
     3901 aactttatca ggatgccacc ggaacaaagc accaaagcga gattctatat ggatattacc
     3961 gtatctgtta tccgtgagct gcccaacgtg agaataaaga taagtcacct gcttgtaaat
     4021 atccataagg ctccgcaatt cccattgcct tttaccattg actttatgag aaagcctctg
     4081 aataaatata tcatattcat cactcttgca tctcttttta ctcttcggat tctttccagt
     4141 tatacgaatg tttccaggga aaatgaaaaa atgaaaatga ggagcaagga taaggcagtc
     4201 ataaatagag tttatttcga tatttagcaa gtccgcgatc ttgtttaaat tgccttcgtt
     4261 gtggtcgtct cttatgtaat tccaaaaagc ttcggagtcg tcattacctt ttaaacctgt
     4321 taacttacgg atagcccgag ttatttccgg cttaatcctg taagggtgaa agattgtagt
     4381 accagcagtt acgccccagc gttccagcct gtcctttaaa ttcctccgga aaagtcggat
     4441 tgcttccaca gatactcccc ggctaaagtc catcgaagca aacccccatg aaggaaactg
     4501 cccagaaaac agggcataag tccacaaaat tacaactaat gcaaatctat gttgcaattc
     4561 cgataggatg ccgcaaccag gacaagaaag tgtattgcag tgagaattaa gatcctttcg
     4621 tgcattataa ttttcagata cagcataagt gaaaggacgc ccacagtaag cgggtctttt
     4681 tccctttgct ggaagaacaa agtcgccaac ttttagagga cgatacttag agcatttaga
     4741 aaaatcgaag tcaggacaag agtagtattt acagatagaa accccgttct ttttgttgcg
     4801 aaccgggcac aaaacaccat cggtacatat acacgtcgca aaatctctac actctggagc
     4861 ataggaaacg gcgccaggtc tagaactaaa atcagaactc agagaacatt tcctgtcgaa
     4921 aatttaaacc gtaaaccaaa gataacaatt ttcataatat tacagaacct atattttttg
     4981 taactcgaca ttacaaaaat gtaccacaaa acataaaaac attttcatag aagaaatatt
     5041 cacaaacaat agaaaaaaat tataagaata agattaacgc ctaccttatt aaaagaaggt
     5101 gtcgagtctt caggttccat caaggcaggc gttgatattc tatatatgtt acacataaca
     5161 tatataacat acgtacctca gaacattcag acctgaatgc tctaaaatga aataagcttt
     5221 aaactttata aattcatctg ctataaatag ttttagagat atagaaagcc caaggaagaa
     5281 aaagaatagc aaaaataaaa aagaaaaacg gcccgcgacg gcaaacccgg agacttataa
     5341 tcaaagtaag acgaaaagga agccgacaac gatttgataa taacaaatca tcttttattt
     5401 tggcaatatt catatgatat atgaagaaat caacatcaag cggattagta tttttgctat
     5461 ttgtaaatca atatcgggtg agacagtata tcaaaaacaa acttcatttt tttcgaacaa
     5521 aaaaatcact atcaaatgac attgtagtaa gaaactacat caaaactaga ggatgattaa
     5581 ttttaagaga gtcttaaaaa atcttcggaa aatagtagta ggtaataaaa aacgccctat
     5641 tcgtaatata ctattcaaaa ctaaatattc attgaaatat ttgggtaata attaatatta
     5701 atctaatatt tagatattga gatataataa taacattttt ttaaaaaatc atctattttc
     5761 atcttaattt tatatattaa agcccatttt ttgagtattc aaattcaaat tattgtgtta
     5821 ttaacatctt atatataaac ttttctattt aatgttaatg aaaaagtgaa tatatataca
     5881 tagagtaatg ttatgatgta tatatcaaaa aaataggagt gattctcatg accgagtaca
     5941 agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc accctcgccg
     6001 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc cacatcgagc
     6061 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     6121 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     6181 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     6241 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     6301 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     6361 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     6421 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgagtgcc
     6481 cgaaggaccg cgcgacctgg tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg
     6541 acccgcagcg cccgaccgaa aggagcgcac gaccccatgg ctccgaccga agccacccgg
     6601 ggcggccccg ccgaccccgc acccgccccc gaggcccacc gcgggggaca caccgaacac
     6661 gccgaccctg ctgaacacgc ggcgcagttc ggtgcccagg agcggatcgg gaattaattc
     6721 gaagctgctg gtgaaagaga ccctatctta cctgctaaaa tctaagttaa ttactaattt
     6781 attattaatt tattattaga ttgggcaaaa tagtaaaaga aaactaaagg aaacctaata
     6841 tggtttcctt tttttatata tttttaattc actgggggca attctgtcag ccgttaagtg
     6901 ttcctgtgtc actgaaaatt gctttgagag gctctaaggg cttctcagtg cgttacatcc
     6961 ctggcttgtt gtccacaacc gttaaacctt aaaagcttta aaagccttat atattctttt
     7021 ttttcttata aaacttaaaa ccttagaggc tatttaagtt gctgatttat attaatttta
     7081 ttgttcaaac atgagagctt agtacgtgaa acatgagagc ttagtacgtt agccatgaga
     7141 gcttagtacg ttagccatga gggtttagtt cgttaaacat gagagcttag tacgttaaac
     7201 atgagagctt agtacgtgaa acatgagagc ttagtacgta ctatcaacag gttgaactgc
     7261 tgatcttcag atcctctacg ccggacgcat cgtggggatc taaaaaaaag cccgctcatt
     7321 aggcgggctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     7381 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     7441 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     7501 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     7561 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     7621 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     7681 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     7741 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     7801 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7861 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7921 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     7981 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     8041 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     8101 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     8161 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     8221 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     8281 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacct
//
