LOCUS       Exported               17407 bp ds-DNA     circular SYN 13-JAN-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17407)
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..17407
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(145..516)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /note="traJ"
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            549..658
                     /note="incP origin of transfer"
     CDS             complement(1339..1989)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline resistance regulatory protein"
                     /note="TetR"
                     /translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
                     RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
                     PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
                     QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
     misc_feature    2454..2478
                     /note="RB T-DNA repeat"
                     /note="right border repeat from nopaline C58 T-DNA"
     promoter        2602..2781
                     /note="NOS promoter"
                     /note="nopaline synthase promoter"
     promoter        2634..2817
                     /function="NOS promoter"
                     /note="NOS promoter"
                     /note="nopaline synthase promoter"
     CDS             2838..3632
                     /codon_start=1
                     /gene="aph(3)-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="NeoR/KanR"
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     terminator      4022..4274
                     /note="NOS terminator"
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4823..4844
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4859..4889
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    4897..4913
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /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)."
     primer_bind     4921..4937
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5461..5806
                     /note="CaMV 35S promoter"
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     protein_bind    5828..5952
                     /gene="mutant version of attR"
                     /bound_moiety="LR Clonase(TM)"
                     /note="attR1"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        5989..6019
                     /note="lac UV5 promoter"
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             6073..6732
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /note="CmR"
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             7074..7379
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /note="ccdB"
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(7420..7544)
                     /gene="mutant version of attR"
                     /bound_moiety="LR Clonase(TM)"
                     /note="attR2"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             7570..7599
                     /codon_start=1
                     /product="Myc (human c-Myc oncogene) epitope tag"
                     /note="Myc"
                     /translation="EQKLISEEDL"
     CDS             7606..7635
                     /codon_start=1
                     /product="Myc (human c-Myc oncogene) epitope tag"
                     /note="Myc"
                     /translation="EQKLISEEDL"
     CDS             7651..7680
                     /codon_start=1
                     /product="Myc (human c-Myc oncogene) epitope tag"
                     /note="Myc"
                     /translation="EQKLISEEDL"
     CDS             7687..7716
                     /codon_start=1
                     /product="Myc (human c-Myc oncogene) epitope tag"
                     /note="Myc"
                     /translation="EQKLISEEDL"
     terminator      7747..7999
                     /note="NOS terminator"
                     /note="nopaline synthase terminator and poly(A) signal"
     terminator      8428..8680
                     /note="NOS terminator"
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(9148..10173)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /note="HygR"
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(10183..10527)
                     /note="CaMV 35S promoter"
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     complement(10637..10653)
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    11271..11295
                     /note="LB T-DNA repeat"
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             complement(12891..14039)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /note="trfA"
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     CDS             complement(14338..15132)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /note="KanR"
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     mobile_element  15324..16091
                     /mobile_element_type="insertion sequence:IS1"
                     /note="IS1"
                     /note="prokaryotic transposable element"
     rep_origin      16714..18
                     /note="oriV"
                     /note="incP origin of replication"