LOCUS       Exported                7019 bp DNA     circular SYN 30-MAY-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 7019)
  AUTHORS   Camilla Ciolli Mattioli
  TITLE     Direct Submission
  JOURNAL   Exported Jun 7, 2022 from SnapGene 6.0.5
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7019
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     mobile_element  152..317
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     primer_bind     413..454
                     /label=129_pSA_FW
     promoter        458..486
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    494..510
                     /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)."
     CDS             533..1249
                     /codon_start=1
                     /product="Aequoria victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     1295..1313
                     /label=216_lacI_FW
     promoter        1316..1393
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             1394..2476
                     /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"
     primer_bind     complement(2477..2510)
                     /label=131_pSA_REV_alternative
     primer_bind     complement(2637..2670)
                     /label=65_for-gibson-in-p5
     protein_bind    complement(2767..2814)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     complement(2814..2835)
                     /label=43_p2_sgRNA-mut_REV
     primer_bind     2871..2890
                     /label=473_kana_FW
     promoter        2891..2919
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             3030..3824
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3084..3103)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3694..3713
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3828..3855)
                     /label=474_kana_REV
     protein_bind    complement(3876..3923)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     terminator      3974..4060
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4163..4257
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(4285..4301)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4286..4302)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     misc_feature    4306..4504
                     /label=Tn7R
     primer_bind     complement(4716..4735)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4835..4857
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4895..4913)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4981..5085
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5086..5946
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5304..5323)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6247..6635
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     oriT            6904..7012
                     /note="incP origin of transfer"
ORIGIN
        1 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcgg gtcagtgagc gaggaagcgg
       61 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagcg
      121 cccaaacata acaggaagaa aaatgccccg ctgtgggcgg acaaaatagt tgggaactgg
      181 gaggggtgga aatggagttt ttaaggatta tttagggaag agtgacaaaa tagatgggaa
      241 ctgggtgtag cgtcgtaagc taatacgaaa attaaaaatg acaaaatagt ttggaactag
      301 atttcactta tctggttctt gaggcggtta aaagagccgt actcttctcc gatgtcgact
      361 aggccatgat gctcattctg tgggaccaaa acgaaaaaac accctttcgg gtgtcttttc
      421 tggaatttgg tactggcaaa tattctgaaa tgagctgttg acaattaatc atcggctcgt
      481 ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaacagaat tcatgcgtaa
      541 aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa
      601 tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac
      661 ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac
      721 tttcggttat ggtgttcaat gctttgcgag atacccagat catatgaaac ggcatgactt
      781 tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga
      841 cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat
      901 cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata
      961 caactataac tcacacaatg tatacatcat ggcagacaaa caaaagaatg gaatcaaagt
     1021 taacttcaaa attagacaca acattgaaga tggaagcgtt caactagcag accattatca
     1081 acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac
     1141 acaatctgcc ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt
     1201 tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaataaa tgtccagacc
     1261 tgcagccaag cttcgacaat tcgcgcgcga aggcgaagcg gcatgcattt acgttgacac
     1321 catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     1381 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     1441 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     1501 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     1561 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     1621 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     1681 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     1741 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     1801 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     1861 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     1921 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     1981 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     2041 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     2101 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     2161 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac
     2221 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
     2281 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     2341 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     2401 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     2461 ggaaagcggg cagtgatctg ttttggcgga tgagttcatg tggctgaccg ttctgttgtc
     2521 tctcgctctt ccgagtagac gaacaataag gcctccctaa cggggggcct tttttattga
     2581 taacaaaagt cagtgcttcc gctatttcca aaataccggg ctaatacggt ttaaacgacc
     2641 tcctggattt gctcagacag ccttttcgtc attcgtttca gccaaaaaac ttaagaccgc
     2701 cggtcttgtc cactaccttg cagtaatgcg gtggacagga tcggcggttt tcttttctct
     2761 tctcaagaag ttcctatact ttctagagaa taggaacttc ggaataggaa cttcctcctg
     2821 aacggccata agaacgaagg ctgtctgttg aactctcgag ccgtggaaac ggatgaaggc
     2881 acgaacccag ttgacataag cctgttcggt tcgtaaactg taatgcaagt agcgtatgcg
     2941 ctcaaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga tcaagatctg
     3001 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3061 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3121 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3181 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3241 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3301 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3361 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3421 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3481 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3541 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3601 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3661 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3721 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3781 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     3841 cgaaatgact cgagcatcag cttcaaaagc gctctgaagt tcctatactt tctagagaat
     3901 aggaacttcg gaataggtac ttcaagatcc ccaattcgag ctcatcgtcc gggccgcaag
     3961 ctcctagcgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac
     4021 gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctcaag ctagagagtc
     4081 attaccccag gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc cccgccctgc
     4141 cactcatcgc agtctagctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg
     4201 ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa
     4261 gctcagctaa ttaagctagc ttatcgatac cgtcgacctc gaaccccacg cccctcttta
     4321 atacgacggg caatttgcac ttcagaaaat gaagagtttg ctttagccat aacaaaagtc
     4381 cagtatgctt tttcacagca taactggact gatttcagtt tacaactatt ctgtctagtt
     4441 taagacttta ttgtcatagt ttagatctat tttgttcagt ttaagacttt attgtccgcc
     4501 cacacctgct tacgcagggc atccatttat tactcaaccg taaccgattt tgccaggtta
     4561 cgcggctggt cctctagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     4621 cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg
     4681 tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4741 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4801 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4861 tcaccgtcgt caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     4921 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     4981 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5041 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5101 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5161 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5221 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5281 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5341 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5401 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5461 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5521 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5581 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5641 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5701 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5761 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtcccgcgg tatcattgca
     5821 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     5881 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     5941 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6001 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6061 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg cagttattgg
     6121 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6181 tgatttaaaa cttcattttt aatttttgcg gccgcaagat ccggccacga tgcgtccggc
     6241 gtagaggatc tgaagatcag cagttcaacc tgttgatagt acgtactaag ctctcatgtt
     6301 tcacgtacta agctctcatg tttaacgtac taagctctca tgtttaacga actaaaccct
     6361 catggctaac gtactaagct ctcatggcta acgtactaag ctctcatgtt tcacgtacta
     6421 agctctcatg tttgaacaat aaaattaata taaatcagca acttaaatag cctctaaggt
     6481 tttaagtttt ataagaaaaa aaagaatata taaggctttt aaagctttta aggtttaacg
     6541 gttgtggaca acaagccagg gatgtaacgc actgagaagc ccttagagcc tctcaaagca
     6601 attttgagtg acacaggaac acttaacggc tgacatggga attagccatg gcatcacagt
     6661 atcgtgatga cagaggcagg gagtgcggcc tttttacggt tcctggcctt ttgctggcct
     6721 tttgctcaca tgttctttcc tgcgttatca ggggattcct taaggtatac tttccgctgc
     6781 ataaccctgc ttcggggtca ttatagcgat tttttcggta tatccatcct ttttcgcacg
     6841 atatacagga ttttgccaaa gggttcgtgt agactttcct tggtgtatcc aacggcgtca
     6901 gccgggcagg ataggtgaag taggcccacc cgcgagcggg tgttccttct tcactgtccc
     6961 ttattcgcac ctggcggtgc tcaacgggaa tcctgctctg cgaggctggc cgataagct
//
