LOCUS       Exported                8325 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pAL23, complete sequence.
ACCESSION   MG595924
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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8325
                     /organism="Cloning vector pAL23"
                     /mol_type="other DNA"
                     /db_xref="taxon:2248767"
     misc_feature    1..97
                     /note="truncated pST39 polycistronic multiple cloning site"
     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
     misc_feature    5566..6867
                     /label=pMip1 integration vector sequence (P. Gernhardt)
                     /note="pMip1 integration vector sequence (P. Gernhardt)"
     regulatory      5566..5927
                     /regulatory_class="promoter"
                     /note="mcr promoter from Methanococcus voltae"
     CDS             5928..6527
                     /codon_start=1
                     /gene="pac"
                     /product="puromycin transacetylase"
                     /label=pac
                     /note="confers puromycin resistance"
                     /protein_id="AWW92031.1"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     gene            5928..6527
                     /gene="pac"
                     /label=pac
     regulatory      6528..6867
                     /regulatory_class="terminator"
                     /note="mcr terminator from Methanococcus voltae"
     rep_origin      6885..7273
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     misc_feature    6885..7262
                     /label=pGP704 suicide vector sequence (G. Pearson)
                     /note="pGP704 suicide vector sequence (G. Pearson)"
     rep_origin      complement(6885..7260)
                     /direction=LEFT
                     /label=r6k ori
                     /note="r6k ori"
     regulatory      complement(7302..7334)
                     /regulatory_class="terminator"
                     /note="bla terminator"
     misc_feature    7328..8302
                     /note="pBluescript KS(+) cloning vector sequence 
                     (Stratagene)"
     CDS             complement(7335..8195)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /note="confers ampicillin resistance"
                     /protein_id="AWW92032.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(7335..8195)
                     /gene="bla"
                     /label=bla
     regulatory      complement(8196..8300)
                     /regulatory_class="promoter"
                     /note="bla promoter"
     promoter        complement(8196..8300)
                     /gene="bla"
                     /label=AmpR promoter