LOCUS       Exported                7552 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 7552)
  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..7552
                     /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             580..1293
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     promoter        1402..1479
                     /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             1480..2562
                     /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"
     terminator      2808..2894
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2986..3013
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3007..3043)
                     /label=130_pSA_REV
     primer_bind     complement(3170..3203)
                     /label=65_for-gibson-in-p5
     protein_bind    complement(3300..3347)
                     /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(3347..3368)
                     /label=43_p2_sgRNA-mut_REV
     primer_bind     3404..3423
                     /label=473_kana_FW
     promoter        3424..3452
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             3563..4357
                     /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(3617..3636)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4227..4246
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4361..4388)
                     /label=474_kana_REV
     protein_bind    complement(4409..4456)
                     /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      4507..4593
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4696..4790
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(4818..4834)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4819..4835)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     misc_feature    4839..5037
                     /label=Tn7R
     primer_bind     complement(5249..5268)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5368..5390
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5428..5446)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5514..5618
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5619..6479
                     /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(5837..5856)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6780..7168
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     oriT            7437..7545
                     /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 tcgagctcgg
      541 tacccgggga tcctctagat ttaagaagga gatatacata tgagtaaagg agaagaactt
      601 ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt
      661 tctgtcagtg gagagggtga aggtgatgca acatacggaa aacttaccct taaatttatt
      721 tgcactactg gaaaactacc tgttccatgg ccaacacttg tcactacttt cggttatggt
      781 gttcaatgct ttgcgagata cccagatcat atgaaacagc atgacttttt caagagtgcc
      841 atgcccgaag gttatgtaca ggaaagaact atatttttca aagatgacgg gaactacaag
      901 acacgtgctg aagtcaagtt tgaaggtgat acccttgtta atagaatcga gttaaaaggt
      961 attgatttta aagaagatgg aaacattctt ggacacaaat tggaatacaa ctataactca
     1021 cacaatgtat acatcatggc agacaaacaa aagaatggaa tcaaagttaa cttcaaaatt
     1081 agacacaaca ttgaagatgg aagcgttcaa ctagcagacc attatcaaca aaatactcca
     1141 attggcgatg gccctgtcct tttaccagac aaccattacc tgtccacaca atctgccctt
     1201 tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct
     1261 gggattacac atggcatgga tgaactatac aaaaggcctg cagcaaacga cgaaaactac
     1321 gctgcagcag tttaaatgtc cagacctgca gccaagcttc gacaattcgc gcgcgaaggc
     1381 gaagcggcat gcatttacgt tgacaccatc gaatggtgca aaacctttcg cggtatggca
     1441 tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac
     1501 gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc
     1561 agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac
     1621 attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc
     1681 acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc
     1741 gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt
     1801 aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg
     1861 ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt
     1921 cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg
     1981 cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc
     2041 ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc
     2101 aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa
     2161 caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat
     2221 cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat
     2281 atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc
     2341 accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc
     2401 tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa
     2461 accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     2521 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     2581 aagttagcgc gaattgtcga agcttggctg ttttggcgga tgagagaaga ttttcagcct
     2641 gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag
     2701 tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga
     2761 tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa
     2821 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
     2881 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt
     2941 ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga
     3001 cggatggcct ttttgcgttt ctacaaattc atgtggctga ccgttctgtt gtctctcgct
     3061 cttccgagta gacgaacaat aaggcctccc taacgggggg ccttttttat tgataacaaa
     3121 agtcagtgct tccgctattt ccaaaatacc gggctaatac ggtttaaacg acctcctgga
     3181 tttgctcaga cagccttttc gtcattcgtt tcagccaaaa aacttaagac cgccggtctt
     3241 gtccactacc ttgcagtaat gcggtggaca ggatcggcgg ttttcttttc tcttctcaag
     3301 aagttcctat actttctaga gaataggaac ttcggaatag gaacttcctc ctgaacggcc
     3361 ataagaacga aggctgtctg ttgaactctc gagccgtgga aacggatgaa ggcacgaacc
     3421 cagttgacat aagcctgttc ggttcgtaaa ctgtaatgca agtagcgtat gcgctcaaaa
     3481 ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagat ctgatcaaga
     3541 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3601 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3661 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3721 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     3781 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     3841 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     3901 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     3961 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4021 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4081 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4141 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4201 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4261 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4321 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4381 actcgagcat cagcttcaaa agcgctctga agttcctata ctttctagag aataggaact
     4441 tcggaatagg tacttcaaga tccccaattc gagctcatcg tccgggccgc aagctcctag
     4501 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
     4561 ccagtctttc gactgagcct ttcgttttat ttgatgcctc aagctagaga gtcattaccc
     4621 caggcgttta agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcat
     4681 cgcagtctag cttggattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa
     4741 caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc caagctcagc
     4801 taattaagct agcttatcga taccgtcgac ctcgaacccc acgcccctct ttaatacgac
     4861 gggcaatttg cacttcagaa aatgaagagt ttgctttagc cataacaaaa gtccagtatg
     4921 ctttttcaca gcataactgg actgatttca gtttacaact attctgtcta gtttaagact
     4981 ttattgtcat agtttagatc tattttgttc agtttaagac tttattgtcc gcccacacct
     5041 gcttacgcag ggcatccatt tattactcaa ccgtaaccga ttttgccagg ttacgcggct
     5101 ggtcctctag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     5161 gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     5221 tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     5281 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5341 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5401 cgtcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     5461 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     5521 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5581 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5641 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5701 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5761 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5821 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5881 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5941 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6001 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6061 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6121 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6181 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6241 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6301 ttattgctga taaatctgga gccggtgagc gtgggtcccg cggtatcatt gcagcactgg
     6361 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6421 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6481 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6541 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6601 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggcagttat tggtgcctca
     6661 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6721 aaacttcatt tttaattttt gcggccgcaa gatccggcca cgatgcgtcc ggcgtagagg
     6781 atctgaagat cagcagttca acctgttgat agtacgtact aagctctcat gtttcacgta
     6841 ctaagctctc atgtttaacg tactaagctc tcatgtttaa cgaactaaac cctcatggct
     6901 aacgtactaa gctctcatgg ctaacgtact aagctctcat gtttcacgta ctaagctctc
     6961 atgtttgaac aataaaatta atataaatca gcaacttaaa tagcctctaa ggttttaagt
     7021 tttataagaa aaaaaagaat atataaggct tttaaagctt ttaaggttta acggttgtgg
     7081 acaacaagcc agggatgtaa cgcactgaga agcccttaga gcctctcaaa gcaattttga
     7141 gtgacacagg aacacttaac ggctgacatg ggaattagcc atggcatcac agtatcgtga
     7201 tgacagaggc agggagtgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7261 acatgttctt tcctgcgtta tcaggggatt ccttaaggta tactttccgc tgcataaccc
     7321 tgcttcgggg tcattatagc gattttttcg gtatatccat cctttttcgc acgatataca
     7381 ggattttgcc aaagggttcg tgtagacttt ccttggtgta tccaacggcg tcagccgggc
     7441 aggataggtg aagtaggccc acccgcgagc gggtgttcct tcttcactgt cccttattcg
     7501 cacctggcgg tgctcaacgg gaatcctgct ctgcgaggct ggccgataag ct
//
