LOCUS       Exported               13905 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pGRG36pir, complete sequence.
ACCESSION   JX239260
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13905)
  AUTHORS   Kvitko BH, Bruckbauer S, Prucha J, McMillan I, Breland EJ, Lehman S,
            Mladinich K, Choi KH, Karkhoff-Schweizer R, Schweizer HP.
  TITLE     A simple method for construction of pir+ Enterobacterial hosts for 
            maintenance of R6K replicon plasmids
  JOURNAL   BMC Res Notes 5, 157 (2012)
  PUBMED    22433797
REFERENCE   2  (bases 1 to 13905)
  AUTHORS   Bruckbauer ST.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-JUN-2012) Microbiology Immunology and Pathology, 
            Colorado State University, IDRC at Foothills Campus, Campus Delivery
            0922, Fort Collins, CO 80523-0922, USA
REFERENCE   3  (bases 1 to 13905)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..13905
                     /organism="Cloning vector pGRG36pir"
                     /mol_type="other DNA"
                     /db_xref="taxon:1227933"
     CDS             complement(146..1075)
                     /codon_start=1
                     /gene="araC"
                     /product="AraC"
                     /label=araC
                     /note="Activates transcription of Tns proteins in the 
                     presence of L-arabinose, inhibits the transcription of Tns 
                     proteins without L-arabinose."
                     /protein_id="AFR24694.1"
                     /translation="MQYGQLVSSLNGGSMKSMAEAQNDPLLPGYSFNAHLVAGLTPIEA
                     NGYLDFFIDRPLGMKGYILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPE
                     AREWYHQWVYFRPRAYWHEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGR
                     YSELLAINLLEQLLLRRMEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVC
                     LSPSRLSHLFRQQLGISVLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRV
                     FKKCTGASPSEFRAGCEEKVNDVAVKLS"
     gene            complement(146..1075)
                     /gene="araC"
                     /label=araC
     CDS             complement(146..1024)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1051..1335
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     CDS             1453..2274
                     /codon_start=1
                     /gene="tnsA"
                     /product="TnsA"
                     /label=tnsA
                     /note="Tn7 transposase A"
                     /protein_id="AFR24690.1"
                     /translation="MAKANSSFSEVQIARRIKEGRGQGHGKDYIPWLTVQEVPSSGRSH
                     RIYSHKTGRVHHLLSDLELAVFLSLEWESSVLDIREQFPLLPSDTRQIAIDSGIKHPVI
                     RGVDQVMSTDFLVDCKDGPFEQFAIQVKPAAALQDERTLEKLELERRYWQQKQIPWFIF
                     TDKEINPVVKENIEWLYSVKTEEVSAELLAQLSPLAHILQEKGDENIINVCKQVDIAYD
                     LELGKTLSEIRALTANGFIKFNIYKSFRANKCADLCISQVVNMEELRYVAN"
     gene            1453..2274
                     /gene="tnsA"
                     /label=tnsA
     CDS             2261..4369
                     /codon_start=1
                     /gene="tnsB"
                     /product="TnsB"
                     /label=tnsB
                     /note="Tn7 transposase B"
                     /protein_id="AFR24691.1"
                     /translation="MWQINEVVLFDNDPYRILAIEDGQVVWMQISADKGVPQARAELLL
                     MQYLDEGRLVRTDDPYVHLDLEEPSVDSVSFQKREEDYRKILPIINSKDRFDPKVRSEL
                     VEHVVQEHKVTKATVYKLLRRYWQRGQTPNALIPDYKNSGAPGERRSATGTAKIGRARE
                     YGKGEGTKVTPEIERLFRLTIEKHLLNQKGTKTTVAYRRFVDLFAQYFPRIPQEDYPTL
                     RQFRYFYDREYPKAQRLKSRVKAGVYKKDVRPLSSTATSQALGPGSRYEIDATIADIYL
                     VDHHDRQKIIGRPTLYIVIDVFSRMITGFYIGFENPSYVVAMQAFVNACSDKTAICAQH
                     DIEISSSDWPCVGLPDVLLADRGELMSHQVEALVSSFNVRVESAPPRRGDAKGIVESTF
                     RTLQAEFKSFAPGIVEGSRIKSHGETDYRLDASLSVFEFTQIILRTILFRNNHLVMDKY
                     DRDADFPTDLPSIPVQLWQWGMQHRTGSLRAVEQEQLRVALLPRRKVSISSFGVNLWGL
                     YYSGSEILREGWLQRSTDIARPQHLEAAYDPVLVDTIYLFPQVGSRVFWRCNLTERSRQ
                     FKGLSFWEVWDIQAQEKHNKANAKQDELTKRRELEAFIQQTIQKANKLTPSTTEPKSTR
                     IKQIKTNKKEAVTSERKKRAEHLKPSSSGDEAKVIPFNAVEADDQEDYSLPTYVPELFQ
                     DPPEKDES"
     gene            2261..4369
                     /gene="tnsB"
                     /label=tnsB
     CDS             4366..6033
                     /codon_start=1
                     /gene="tnsC"
                     /product="TnsC"
                     /label=tnsC
                     /note="Tn7 transposase C"
                     /protein_id="AFR24692.1"
                     /translation="MSATRIQAVYRDTGVEAYRDNPFIEALPPLQESVNSAASLKSSLQ
                     LTSSDLQKSRVIRAHTICRIPDDYFQPLGTHLLLSERISVMIRGGYVGRNPKTGDLQKH
                     LQNGYERVQTGELETFRFEEARSTAQSLLLIGCSGSGKTTSLHRILATYPQVIYHRELN
                     VEQVVYLKIDCSHNGSLKEICLNFFRALDRALGSNYERRYGLKRHGIETMLALMSQIAN
                     AHALGLLVIDEIQHLSRSRSGGSQEMLNFFVTMVNIIGVPVMLIGTPKAREIFEADLRS
                     ARRGAGFGAIFWDPIQQTQRGKPNQEWIAFTDNLWQLQLLQRKDALLSDEVRDVWYELS
                     QGVMDIVVKLFVLAQLRALALGNERITAGLLRQVYQDELKPVHPMLEALRSGIPERIAR
                     YSDLVVPEIDKRLIQLQLDIAAIQEQTPEEKALQELDTEDQRHLYLMLKEDYDSSLLIP
                     TIKKAFSQNPTMTRQKLLPLVLQWLMEGETVVSELEKPSKSKKVSAIKVVKPSDWDSLP
                     DTDLRYIYSQRQPEKTMHERLKGKGVIVDMASLFKQAG"
     gene            4366..6033
                     /gene="tnsC"
                     /label=tnsC
     CDS             6036..7562
                     /codon_start=1
                     /gene="tnsD"
                     /product="TnsD"
                     /label=tnsD
                     /note="Tn7 transposase D"
                     /protein_id="AFR24693.1"
                     /translation="MRNFPVPYSNELIYSTIARAGVYQGIVSPKQLLDEVYGNRKVVAT
                     LGLPSHLGVIARHLHQTGRYAVQQLIYEHTLFPLYAPFVGKERRDEAIRLMEYQAQGAV
                     HLMLGVAASRVKSDNRFRYCPDCVALQLNRYGEAFWQRDWYLPALPYCPKHGALVFFDR
                     AVDDHRHQFWALGHTELLSDYPKDSLSQLTALAAYIAPLLDAPRAQELSPSLEQWTLFY
                     QRLAQDLGLTKSKHIRHDLVAERVRQTFSDEALEKLDLKLAENKDTCWLKSIFRKHRKA
                     FSYLQHSIVWQALLPKLTVIEALQQASALTEHSITTRPVSQSVQPNSEDLSVKHKDWQQ
                     LVHKYQGIKAARQSLEGGVLYAWLYRHDRDWLVHWNQQHQQERLAPAPRVDWNQRDRIA
                     VRQLLRIIKRLDSSLDHPRATSSWLLKQTPNGTSLAKNLQKLPLVALCLKRYSESVEDY
                     QIRRISQAFIKLKQEDVELRRWRLLRSATLSKERITEEAQRFLEMVYGEE"
     gene            6036..7562
                     /gene="tnsD"
                     /label=tnsD
     terminator      8196..8282
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      8374..8401
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        8420..8511
                     /gene="bla"
                     /label=AmpR promoter
     CDS             8512..9372
                     /codon_start=1
                     /gene="bla"
                     /product="Beta Lactamase"
                     /label=bla
                     /note="Beta Lactamase conferring ampicillin resistance"
                     /protein_id="AFR24695.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            8512..9372
                     /gene="bla"
                     /label=bla
     oriT            9543..9652
                     /note="incP origin of transfer"
     misc_feature    9555..9641
                     /label=oriT
                     /note="oriT"
     rep_origin      10392..10614
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     rep_origin      10661..12030
                     /label=pSC101-ori-ts
                     /note="pSC101-ori-ts"
     CDS             10662..11612
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     misc_feature    12045..12276
                     /label=Tn7L
                     /note="Tn7L"
     mobile_element  12045..12210
                     /mobile_element_type="transposon:Tn7"
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     misc_recomb     12313..12337
                     /label=attB1
                     /note="attB1"
     protein_bind    12313..12337
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             12472..13389
                     /codon_start=1
                     /gene="pir"
                     /product="Pi"
                     /label=pir
                     /note="Pi protein replicates vectors with an R6K origin"
                     /protein_id="AFR24696.1"
                     /translation="MRLKVMMDVNKKTKIRHRNELNHTLAQLPLPAKRVMYMALAPIDS
                     KEPLERGRVFKIRAEDLAALAKITPSLAYRQLKEGGKLLGASKISLRGDDIIASAKELN
                     LLFTAKDSPEELDLNIIEWIAYSNDEGYLSLKFTRTIEPYISSLIGKKNKFTTQLLTAS
                     LRLSSQYSSSLYQLIRKHYSNFKKKNYFIISVDELKEELIAYTFDKDGSIEYKYPDFPI
                     FKRDVLNKAIAEIKKKTEISFVGFTVHEKEGRKISKLKFEFVVDEDEFSGDKDDEAFFM
                     NLSEANAAFLKVFDETVPPKKAKG"
     gene            12472..13389
                     /gene="pir"
                     /label=pir
     misc_recomb     13603..13627
                     /label=attB2
                     /note="attB2"
     protein_bind    complement(13603..13627)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    13694..13905
                     /label=Tn7R
                     /note="Tn7R"