LOCUS       Exported               11418 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Reporter vector pAL36, complete sequence.
ACCESSION   MG595925
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11418)
  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 11418)
  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 11418)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..11418
                     /organism="Reporter vector pAL36"
                     /mol_type="other DNA"
                     /db_xref="taxon:2248768"
     regulatory      7..90
                     /regulatory_class="promoter"
                     /note="minimal mcrB promoter with tetracycline operator"
     protein_bind    46..63
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      91..109
                     /regulatory_class="ribosome_binding_site"
                     /note="mcrB RBS from Methanosarcina acetivorans C2A"
     CDS             110..3184
                     /codon_start=1
                     /gene="lacZ"
                     /product="beta-galactosidase"
                     /label=lacZ
                     /protein_id="AWW92033.1"
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRWVGYG
                     QDSRLPSEFDLSAFLRAGENRLAVMVLRWSDGSYLEDQDMWRMSGIFRDVSLLHKPTTQ
                     ISDFHVATRFNDDFSRAVLEAEVQMCGELRDYLRVTVSLWQGETQVASGTAPFGGEIID
                     ERGGYADRVTLRLNVENPKLWSAEIPNLYRAVVELHTADGTLIEAEACDVGFREVRIEN
                     GLLLLNGKPLLIRGVNRHEHHPLHGQVMDEQTMVQDILLMKQNNFNAVRCSHYPNHPLW
                     YTLCDRYGLYVVDEANIETHGMVPMNRLTDDPRWLPAMSERVTRMVQRDRNHPSVIIWS
                     LGNESGHGANHDALYRWIKSVDPSRPVQYEGGGADTTATDIICPMYARVDEDQPFPAVP
                     KWSIKKWLSLPGETRPLILCEYAHAMGNSLGGFAKYWQAFRQYPRLQGGFVWDWVDQSL
                     IKYDENGNPWSAYGGDFGDTPNDRQFCMNGLVFADRTPHPALTEAKHQQQFFQFRLSGQ
                     TIEVTSEYLFRHSDNELLHWMVALDGKPLASGEVPLDVAPQGKQLIELPELPQPESAGQ
                     LWLTVRVVQPNATAWSEAGHISAWQQWRLAENLSVTLPAASHAIPHLTTSEMDFCIELG
                     NKRWQFNRQSGFLSQMWIGDKKQLLTPLRDQFTRAPLDNDIGVSEATRIDPNAWVERWK
                     AAGHYQAEAALLQCTADTLADAVLITTAHAWQHQGKTLFISRKTYRIDGSGQMAITVDV
                     EVASDTPHPARIGLNCQLAQVAERVNWLGLGPQENYPDRLTAACFDRWDLPLSDMYTPY
                     VFPSENGLRCGTRELNYGPHQWRGDFQFNISRYSQQQLMETSHRHLLHAEEGTWLNIDG
                     FHMGIGGDDSWSPSVSAEFQLSAGRYHYQLVWCQK"
     gene            110..3184
                     /gene="lacZ"
                     /label=lacZ
     primer_bind     complement(130..146)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3192..8662
                     /label=pC2A sequence
                     /note="pC2A sequence"
     CDS             complement(3798..4742)
                     /codon_start=1
                     /gene="ssrA"
                     /product="site-specific recombinase A"
                     /label=ssrA
                     /protein_id="AWW92034.1"
                     /translation="MGEMAAIQLTGIYEPLDNERLISLFTSDCIGRGYSKRTIEGYVSH
                     AKFFLQMCGVNAGFPELEGFLVHIRDERQYTLATCNNYFASLSTFFDFLEFKHIIERNN
                     IPTFRKRYLRSYKKYHTPETRQLITIEDMAKLVKVPRYPMYQALIIFLAKTGIRRNELI
                     TLDRADIDLEKCTVRLKKTAKRSNRIVFFDLETKAFLEAYLNTRHDKEKALFIGRQNGH
                     RVQRNLVYDIVTSAASSIGLHNPEGRLEEHFTPHCCRHWFTTWLRRSGMERTFIQELRG
                     DSRGEAIDVYDHIEKDELREAYLKCIPQLGIKP"
     gene            complement(3798..4742)
                     /gene="ssrA"
                     /label=ssrA
     misc_feature    5225..5872
                     /label=Orf2
                     /note="Orf2"
     misc_feature    complement(5878..6306)
                     /label=Orf1
                     /note="Orf1"
     CDS             complement(6385..7995)
                     /codon_start=1
                     /gene="repA"
                     /product="regulatory protein RepA"
                     /label=repA
                     /protein_id="AWW92035.1"
                     /translation="MSSDFSSRPGAVSYAPECRDFATCICTDGVLCPVRNKKNGVSICK
                     YYSCPDFDFSKCSKYRPLKVGDFVLPAKGKRPAYCGRPFTYAVSENYNARKDLNSHCNT
                     LSCPGCGILSELQHRFALVVILWTYALFSGQFPSWGFASMDFSRGVSVEAIRLFRRNLK
                     DRLERWGVTAGTTIFHPYRIKPEITRAIRKLTGLKGNDDSEAFWNYIRDDHNEGNLNKI
                     ADLLNIEINSIYDCLILAPHFHFFIFPGNIRITGKNPKSKKRCKSDEYDIFIQRLSHKV
                     NGKRQWELRSLMDIYKQVTYLYSHVGQLTDNRYGNIHIESRFGALFRWHPDKVVSSSGK
                     LTFEQLIDTRTEVLNLINEGREVKLVYNGELKWEGQENDDTGWIPLTKLRDHFSGGRQA
                     LHAYLKEAKAVKPEYADYLQQLIARNNFYHNLKGRDGKNKVPKKWRKLWASPFKVDELP
                     EFLKLEPAGSLLRVWFLEGLPDPPAGFLVVDSHQWEHDGCIIEESPEMPSNSLQKASEI
                     NYEELFTSLKSRSMMFGGD"
     gene            complement(6385..7995)
                     /gene="repA"
                     /label=repA
     misc_feature    8659..9960
                     /label=pMip1 integration vector sequence (P. Gernhardt)
                     /note="pMip1 integration vector sequence (P. Gernhardt)"
     regulatory      8659..9020
                     /regulatory_class="promoter"
                     /note="mcr promoter from Methanococcus voltae"
     CDS             9021..9620
                     /codon_start=1
                     /gene="pac"
                     /product="puromycin transacetylase"
                     /label=pac
                     /note="confers puromycin resistance"
                     /protein_id="AWW92036.1"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     gene            9021..9620
                     /gene="pac"
                     /label=pac
     regulatory      9621..9960
                     /regulatory_class="terminator"
                     /note="mcr terminator from Methanococcus voltae"
     rep_origin      9978..10366
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     misc_feature    9978..10355
                     /label=pGP704 suicide vector sequence (G. Pearson)
                     /note="pGP704 suicide vector sequence (G. Pearson)"
     rep_origin      complement(9978..10353)
                     /direction=LEFT
                     /label=r6k ori
                     /note="r6k ori"
     regulatory      complement(10395..10427)
                     /regulatory_class="terminator"
                     /note="bla terminator"
     misc_feature    10421..11413
                     /note="pBluescript KS(+) cloning vector sequence 
                     (Stratagene)"
     CDS             complement(10428..11288)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /note="confers ampicillin resistance"
                     /protein_id="AWW92037.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(10428..11288)
                     /gene="bla"
                     /label=bla
     regulatory      complement(11289..11393)
                     /regulatory_class="promoter"
                     /note="bla promoter"
     promoter        complement(11289..11393)
                     /gene="bla"
                     /label=AmpR promoter