LOCUS       Exported                6900 bp ds-DNA     circular SYN 06-MAR-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6900)
  AUTHORS   Manuel Iburg
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Jul 18, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6900
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      13..468
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(478..497)
                     /label=148 SpecRpCDF rev
     CDS             complement(561..1352)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1459..2277
                     /direction=RIGHT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     rep_origin      1474..1488
                     /direction=RIGHT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     rep_origin      1572..1636
                     /direction=RIGHT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     2382..2403
                     /label=148 SpecRpCDF for
     misc_feature    2463..2602
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2704..2895)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3704..4786)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(4787..4864)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        5173..5191
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5192..5216
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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     complement(5217..5299)
                     /label=Vec rev LgNterm
     primer_bind     complement(5223..5260)
                     /label=pET26-Gib2-R
                     /note="Ta = 62.5Tm = 61.5"
     primer_bind     complement(5223..5260)
                     /label=pET26b-OptHis-Gib-R
                     /note="Ta = 63.4Tm = 60.4"
     primer_bind     complement(5234..5266)
                     /label=p26-5HisMoLinker-R
                     /note="Ta = 60.4Tm = 57.4"
     primer_bind     complement(5240..5275)
                     /label=FLAG-LgBit rev
     CDS             5264..5287
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     5276..5308
                     /label=FLAG-LgBit for
     CDS             5288..5758
                     /codon_start=1
                     /label=NanoBiT Large Fragment
                     /note="https://www.promega.com/products/protein-purificatio
                     n-and-interactions/protein-interactions/nanobit-complementa
                     tion-assay-for-protein-interactions/nanobit-ppi-starter-sys
                     tems/?activeTab=0"
                     /translation="VFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRI
                     VRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVT
                     PNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTIN"
     primer_bind     5288..5310
                     /label=F1 for LgNterm
     primer_bind     5288..5308
                     /label=T-Beast-11S-IF
                     /note="Ta =59.5Tm = 58.5"
     primer_bind     complement(5736..5768)
                     /label=F1 rev LgNterm
     primer_bind     5750..5779
                     /label=Vec for LgNterm
     CDS             5752..5758
                     /codon_start=1
                     /product="Binds to Rapamycin analogue (rapalogue) AP21967 
                     as well as to Rapamycin. "
                     /label=FRB (T2098L)
                     /translation="IN"
     primer_bind     5759..5777
                     /label=T-NLink-11S-IF
                     /note="Ta = 59.5Tm = 58.5"
     primer_bind     5759..5777
                     /label=11S-Swap-VF
                     /note="Ta = 61.5Tm = 58.7"
     primer_bind     complement(5798..5820)
                     /label=Vec Luc-N r
     primer_bind     5806..5841
                     /label=Luc-N f
     CDS             5813..6736
                     /codon_start=1
                     /label=GvpN
                     /translation="TVLTDKRKKGSGAFIQDDETKEVLSRALSYLKSGYSIHFTGPAGG
                     GKTSLARALAKKRKRPVMLMHGNHELNNKDLIGDFTGYTSKKVIDQYVRSVYKKDEQVS
                     ENWQDGRLLEAVKNGYTLIYDEFTRSKPATNNIFLSILEEGVLPLYGVKMTDPFVRVHP
                     DFRVIFTSNPAEYAGVYDTQDALLDRLITMFIDYKDIDRETAILTEKTDVEEDEARTIV
                     TLVANVRNRSGDENSSGLSLRASLMIATLATQQDIPIDGSDEDFQTLCIDILHHPLTKC
                     LDEENAKSKAEKIILEECKNIDTEEK"
     primer_bind     5840..5865
                     /label=Bm1.1seqF
     misc_feature    5872
                     /label=SNP
                     /note="Bacillus megaterium QM B1551 SNP"
     primer_bind     complement(5910..5925)
                     /label=GvpN_rv1
     primer_bind     6242..6261
                     /label=Bm2seqF
     primer_bind     complement(6688..6746)
                     /label=Luc-N r
     primer_bind     6727..6753
                     /label=Vec Luc-N f
     primer_bind     6737..6754
                     /label=pBAD-ddXFP-VF
                     /note="Ta = 62.6Tm = 61.6"
     primer_bind     complement(6741..6763)
                     /label=rrnB-F1060_Seq-R
     primer_bind     complement(6743..6782)
                     /label=pBAD2pETv2
                     /note="Ta = 64Tm = 63"
     primer_bind     6763..6784
                     /label=pET26b-ChangeHis-F
                     /note="Ta = 68Tm = 65"
     primer_bind     6763..6780
                     /label=pET26-Gib2-F
                     /note="Ta = 62.5Tm = 64"
     primer_bind     6763..6780
                     /label=p26-5HisMoLinker-F
                     /note="Ta = 60.4Tm = 62"
     terminator      6829..6876
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
       61 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
      121 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      181 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
      241 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
      301 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
      361 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
      421 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttca ggtggcactt
      481 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
      541 atccgctcat gaattaattc ttatttgccg actaccttgg tgatctcgcc tttcacgtag
      601 tggacaaatt cttccaactg atctgcgcgc gaggccaagc gatcttcttc ttgtccaaga
      661 taagcctgtc tagcttcaag tatgacgggc tgatactggg ccggcaggcg ctccattgcc
      721 cagtcggcag cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta ccaaatgcgg
      781 gacaacgtaa gcactacatt tcgctcatcg ccagcccagt cgggcggcga gttccatagc
      841 gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc aaagagttcc
      901 tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag caagatagcc
      961 agatcaatgt cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt gcgctgccat
     1021 tctccaaatt gcagttcgcg cttagctgga taacgccacg gaatgatgtc gtcgtgcaca
     1081 acaatggtga cttctacagc gcggagaatc tcgctctctc caggggaagc cgaagtttcc
     1141 aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt caccgtaacc
     1201 agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc gtacaaatgt
     1261 acggccagca acgtcggttc gagatggcgc tcgatgacgc caactacctc tgatagttga
     1321 gtcgatactt cggcgatcac cgcttccctc ataacacccc ttgtattact gtttatgtaa
     1381 gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     1441 gtcagacccc gtagaaaaga tcaaaggatc ttcgatcaaa ggatcttctt gagatccttt
     1501 ttttctgcgc gtaatctttt gccctgtaaa cgaaaaaacc acctggggag gtggtttgat
     1561 cgaaggttaa gcagttcccc aactgactta accttcgaaa aaaccacctg gggaggtggt
     1621 ttgatcgaag gttaagtcag ttggggaact gcttaaccgt ggtaactggc tttcgcagag
     1681 cacagcaacc aaatctgtcc ttccagtgta gccggacttt ggcgcacact tcaagagcaa
     1741 ccgcgtgttt agctaaacaa atcctctgcg aactcccagt taccaatggc tgctgccagt
     1801 ggcgttttac cgtgcttttc cgggttggac tcaagtgaac agttaccgga taaggcgcag
     1861 cagtcgggct gaacggggag ttcttgctta cagcccagct tggagcgaac gacctacacc
     1921 gagccgagat accagtgtgt gagctatgag aaagcgccac acttcccgta agggagaaag
     1981 gcggaacagg tatccggtaa acggcagggt cggaacagga gagcgcaaga gggagcgacc
     2041 cgccggaaac ggtggggatc tttaagtcct gtcgggtttc gcccgtactg tcagattcat
     2101 ggttgagcct cacggctccc acagatgcac cggaaaagcg tctgtttatg tgaactctgg
     2161 caggagggcg gagcctatgg aaaaacgcca ccggcgcggc cctgctgttt tgcctcacat
     2221 gttagtcccc tgcttatcca cggaatctgt gggtaacttt gtatgtgtcc gcagcgcaac
     2281 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     2341 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     2401 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     2461 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     2521 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     2581 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     2641 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     2701 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     2761 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     2821 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     2881 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     2941 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     3001 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     3061 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     3121 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     3181 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     3241 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     3301 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     3361 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     3421 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     3481 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     3541 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     3601 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     3661 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     3721 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3781 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     3841 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     3901 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     3961 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     4021 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     4081 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     4141 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     4201 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     4261 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     4321 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     4381 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     4441 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     4501 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     4561 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     4621 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     4681 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     4741 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     4801 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     4861 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     4921 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     4981 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     5041 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     5101 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     5161 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
     5221 tagaaataat tttgtttaac tttaagaagg agatatacat atggattata aagatgatga
     5281 tgataaagtc ttcacactcg aagatttcgt tggggactgg gaacagacag ccgcctacaa
     5341 cctggaccaa gtccttgaac agggaggtgt gtccagtttg ctgcagaatc tcgccgtgtc
     5401 cgtaactccg atccaaagga ttgtccggag cggtgaaaat gccctgaaga tcgacatcca
     5461 tgtcatcatc ccgtatgaag gtctgagcgc cgaccaaatg gcccagatcg aagaggtgtt
     5521 taaggtggtg taccctgtgg atgatcatca ctttaaggtg atcctgccct atggcacact
     5581 ggtaatcgac ggggttacgc cgaacatgct gaactatttc ggacggccgt atgaaggcat
     5641 cgccgtgttc gacggcaaaa agatcactgt aacagggacc ctgtggaacg gcaacaaaat
     5701 tatcgacgag cgcctgatca cccccgacgg ctccatgctg ttccgagtaa ccatcaacgt
     5761 ttctcaaggc agttcaggtg gtggcgggag cggaggtgga ggctcgagcg gtaccgtctt
     5821 aacagacaaa aggaaaaaag gcagtggagc ttttatacaa gatgacgaga caaaagaggt
     5881 tctttcaaga gcgctgagct atttaaaatc cggctattcc attcatttta caggtcctgc
     5941 cggcggaggc aaaacctctt tagcgcgagc gcttgctaaa aagagaaagc gtcctgtaat
     6001 gctgatgcac gggaatcacg agctcaacaa caaagattta attggcgatt ttacgggata
     6061 cacgagcaaa aaagtaatcg accagtacgt tcgttctgtc tataaaaaag atgaacaggt
     6121 gagtgaaaac tggcaggatg gccgattgct tgaagctgta aaaaatggct atacgctgat
     6181 ttacgacgaa tttactcgtt ctaagcctgc gacgaataat atctttctat cgatattaga
     6241 agaaggcgtg ctgccgctgt atggagtaaa aatgaccgat ccttttgtgc gcgtgcatcc
     6301 cgatttccgc gtcatcttca caagcaatcc agctgagtat gccggcgtat atgatacgca
     6361 agatgcgctt ctcgacaggt taattaccat gtttattgat tataaagaca tcgacagaga
     6421 gacagcgatt ttaacggaga aaacggacgt agaagaagat gaagcgcgca caattgtaac
     6481 gctcgtagca aacgtgcgaa accgctctgg agacgaaaac agcagcggac ttagcctgcg
     6541 ggcttcgctt atgatcgcta cccttgccac gcagcaagac attcctatcg atggaagtga
     6601 cgaagatttt caaacgttat gtatcgatat tttgcatcat ccgcttacca aatgtttgga
     6661 tgaagaaaat gcaaaaagca aagccgaaaa aatcatttta gaagaatgta agaatataga
     6721 cactgaagaa aagtaaggct gttttggcgg atgagagaag attgagatcc ggctgctaac
     6781 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     6841 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
//
