LOCUS       Exported                6256 bp DNA     circular SYN 23-MAR-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6256)
  AUTHORS   EJPARKER
  TITLE     Direct Submission
  JOURNAL   Exported Apr 27, 2022 from SnapGene 6.0.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6256
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     repeat_region   274..302
                     /label=IR1
     rep_origin      587..975
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     primer_bind     complement(1350..1377)
                     /label=pHimarEm1+XbaI_RV2
     primer_bind     1357..1399
                     /label=Fragment 1.FOR
     primer_bind     1380..1405
                     /locus_tag=""
                     /label=Geneious name: MCS primer
                     /note="Geneious name: MCS primer"
     primer_bind     1380..1405
                     /label=pFru97 MCS fw
     primer_bind     1380..1403
                     /label=cassette_pAG408overhang_FWD
     primer_bind     1380..1400
                     /label=REV_pFru_ins
     primer_bind     1380..1399
                     /label=cassette5_pGRG36overhang_FWD
     primer_bind     1398..1434
                     /label=71_FWD_Gent
     promoter        1474..1502
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     primer_bind     1474..1502
                     /label=pmre116.for
     primer_bind     1523..1545
                     /label=FWD_Tri
     CDS             1691..2224
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     primer_bind     complement(1691..1708)
                     /label=pAG-GmR-rev
     primer_bind     complement(1710..1723)
                     /label=pAG-GmR-rev
     primer_bind     2201..2218
                     /label=pAG408_rainbowFOR
     primer_bind     complement(2205..2237)
                     /label=72_RV_Gent
     primer_bind     complement(2205..2224)
                     /label=Gent.rev
     primer_bind     complement(2205..2224)
                     /label=pmre113.for
     primer_bind     complement(2207..2224)
                     /label=NotI_GmR_rev
     primer_bind     2225..2240
                     /label=FWD_primer_PnptII
     promoter        2231..2404
                     /locus_tag=""
                     /label=p-nptII promoter
                     /label=p-nptII
     primer_bind     2231..2250
                     /label=11x-nptII-FL-fw
     primer_bind     2231..2245
                     /label=lederm_nptII.fw
     promoter        2380..2567
                     /label=p-nptII promoter
                     /label=p-nptII
     primer_bind     2380..2404
                     /label=Fragment 2.FOR
     primer_bind     complement(2380..2404)
                     /label=pntpII_1_REV
     primer_bind     2380..2404
                     /label=pntpII_1_FWD
     primer_bind     complement(2380..2404)
                     /label=Fragment 1.REV
     primer_bind     2381..2399
                     /label=pFru97xpMRE100_nptIIGib_rv
     primer_bind     complement(2566..2598)
                     /label=REV_primer_PnptII_mTq2/sG2
     primer_bind     complement(2566..2595)
                     /label=REV_primer_PnptII_mCl/mO/mSc/sY2
     primer_bind     complement(2566..2591)
                     /label=REV_primer_PnptII_mTagBFP2
     primer_bind     2576..2608
                     /label=FWD_primer_mTq2/sGFP2
     primer_bind     2576..2605
                     /label=FWD_primer_mCl/mO/mSc/sY2
     primer_bind     2584..2601
                     /label=XhoI_Ptac_XFP_for
     CDS             2586..3302
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     2586..2605
                     /label=FWD_sYFP2
     primer_bind     2586..2603
                     /label=XFPs for
     primer_bind     complement(2586..2603)
                     /label=Ptuf rev
     primer_bind     complement(2631..2652)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3107..3127)
                     /label=FWD_primer_PnptII
     primer_bind     3239..3260
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3253..3272)
                     /label=REV_primer_sG2
     primer_bind     complement(3283..3325)
                     /label=REV_primer_mTq2
     primer_bind     complement(3284..3307)
                     /label=NotI_XFP_rev
     primer_bind     complement(3284..3305)
                     /label=nextgenFP.rv
     primer_bind     complement(3286..3325)
                     /label=REV_primer_mO
     primer_bind     complement(3286..3305)
                     /label=11x-FL-rv
     primer_bind     complement(3287..3325)
                     /label=REV_primer_sY2
     primer_bind     complement(3287..3305)
                     /label=REV_sYFP2
     primer_bind     complement(3287..3305)
                     /label=Ptac+eXFP_rev
     primer_bind     complement(3288..3305)
                     /label=XFPs rev KpnI
     primer_bind     3306..3320
                     /label=Gib_Gent.rev
     primer_bind     complement(3313..3332)
                     /label=FWD_pFru_ins
     primer_bind     3316..3338
                     /label=Gib_Gent.for.cor
     terminator      3322..3416
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(3339..3357)
                     /label=pQE-R
                     /note="19"
     CDS             3460..4119
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3521..3549)
                     /label=pMRE130inserts_rev
     primer_bind     complement(4104..4119)
                     /label=Chloramp_rev
     terminator      4184..4270
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(4275..4292)
                     /label=Gib_Gent.for
     primer_bind     complement(4280..4319)
                     /label=Fragment 2.REV
     primer_bind     complement(4280..4297)
                     /label=pMRE130_cassette2_RV
     primer_bind     complement(4281..4297)
                     /label=cassette_pAG408overhang_RV
     primer_bind     4298..4324
                     /label=pHimarEm1+XmaI_FWD
     repeat_region   4387..4415
                     /label=IR2
     primer_bind     4661..4677
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4757..4768)
                     /label=cassette_pAG408overhang_RV
     oriT            4764..4872
                     /note="incP origin of transfer"
     protein_bind    5019..5040
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5055..5085
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     promoter        5055..5084
                     /gene="lac prom"
                     /label=lac prom promoter
     protein_bind    5093..5109
                     /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)."
     misc_feature    5129..6175
                     /label=Transposase
                     /label=nonstandard type:  ORF
                     /note="/sequence=ORF_2 rf(1)"
ORIGIN
        1 ctagatgcat gctcgagcgg ccgccagtgt gatggatttg aaaaaggaag agtatgagta
       61 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      121 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      181 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      241 gttttccaat gatgagcact tttaaagttc tgctaacagg ttggctgata agtccccggt
      301 ctgacacata gatggcgtcg ctagtattaa atgcatatta tttttatata gtaccaacct
      361 tcaaatgatt cccagatctc aaactggaac aacactcaac cctatctcgg tctattcttt
      421 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
      481 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
      541 ggaaatgtgc gcggaaccag atccccacga tgcgtccggc gtagaggatc tgaagatcag
      601 cagttcaacc tgttgatagt acgtactaag ctctcatgtt tcacgtacta agctctcatg
      661 tttaacgtac taagctctca tgtttaacga actaaaccct catggctaac gtactaagct
      721 ctcatggcta acgtactaag ctctcatgtt tcacgtacta agctctcatg tttgaacaat
      781 aaaattaata taaatcagca acttaaatag cctctaaggt tttaagtttt ataagaaaaa
      841 aaagaatata taaggctttt aaagctttta aggtttaacg gttgtggaca acaagccagg
      901 gatgtaacgc actgagaagc ccttagagcc tctcaaagca attttcagtg acacaggaac
      961 acttaacggc tgacagaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
     1021 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggttaatt
     1081 aagggctgca ggaattcgat atcaagctta tcgataccgt cgaaattctg attaataatt
     1141 tgtttaaatt tttcgtttgg cgtgaggtat ccaagtcttt tacgaggtcg attattgagt
     1201 ttattttcaa tccacttaat ctgtttgttg gttacttcac taaagtcctt accctttggg
     1261 atatactgcc tgataagccc gttggtgttt tcattggcac cacgttccca tgagtggtat
     1321 ggtttgcaaa aatagaattt tatttccaat ttttgcgcaa tttcctcgtg ctctagaata
     1381 taaactgcca ggaattgggg atcggattcg agctttagta cctagattta gatgtctaaa
     1441 aagcggggta ccgaggacgc gtcaattctc gaattgacat aagcctgttc ggttcgtaaa
     1501 ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc
     1561 ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt tgtacagtct
     1621 atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg
     1681 agcagcaacg atgttacgca gcagcaacga tgttacgcag cagggcagtc gccctaaaac
     1741 aaagttaggt ggctcaagta tgggcatcat tcgcacatgt aggctcggcc ctgaccaagt
     1801 caaatccatg cgggctgctc ttgatctttt cggtcgtgag ttcggagacg tagccaccta
     1861 ctcccaacat cagccggact ccgattacct cgggaacttg ctccgtagta agacattcat
     1921 cgcgcttgct gccttcgacc aagaagcggt tgttggcgct ctcgcggctt acgttctgcc
     1981 caagtttgag cagccgcgta gtgagatcta tatctatgat ctcgcagtct ccggcgagca
     2041 ccggaggcag ggcattgcca ccgcgctcat caatctcctc aagcatgagg ccaacgcgct
     2101 tggtgcttat gtgatctacg tgcaagcaga ttacggtgac gatcccgcag tggctctcta
     2161 tacaaagttg ggcatacggg aagaagtgat gcactttgat atcgacccaa gtaccgccac
     2221 ctaaagctcg cacgctgccg caagcactca gggcgcaagg gctgctaaag gaagcggaac
     2281 acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct
     2341 atctggacaa gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca
     2401 tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg
     2461 gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca
     2521 aggatctgat ggcgcagggg atcaagatct gatcaagaga caggatacta gtggaggaag
     2581 aaaaaatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     2641 tggacggcga cgtaaacggc cacaagttca gcgtccgcgg cgagggcgag ggcgatgcca
     2701 ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2761 ccaccctcgt gaccaccttc ggctacggcg tggcctgctt cagccgctac cccgaccaca
     2821 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2881 tctctttcaa ggacgacggt acctacaaga cccgcgccga ggtgaagttc gagggcgaca
     2941 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     3001 ggcacaagct ggagtacaac ttcaacagcc actacgtcta tatcacggcc gacaagcaga
     3061 agaactgcat caaggctaac ttcaagatcc gccacaacgt tgaggacggc agcgtgcagc
     3121 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     3181 accactacct gagccatcag tccaagctga gcaaagaccc caacgagaag cgcgatcaca
     3241 tggtcctgct ggagttcgtg accgccgccg ggattacaca tggcatggac gagctgtaca
     3301 agtaataatt agctgagctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc
     3361 atctggattt gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccaa
     3421 gctagcttgg cgagattttc aggagctaag gaagctaaaa tggagaaaaa aatcactgga
     3481 tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca
     3541 gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc
     3601 gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg
     3661 aatgctcatc cggaatttcg tatggcaatg aaagacggtg agctggtgat atgggatagt
     3721 gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc gctctggagt
     3781 gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt ggcgtgttac
     3841 ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt cgtctcagcc
     3901 aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga caacttcttc
     3961 gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg
     4021 gcgattcagg ttcatcatgc cgtctgtgat ggcttccatg tcggcagaat gcttaatgaa
     4081 ttacaacagt actgcgatga gtggcagggc ggggcgtaat ttttttaagg cagttattgg
     4141 tgcccttaaa cgcctggggt aatgactctc tagcttgagg catcaaataa aacgaaaggc
     4201 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     4261 taggacaaat ccgccgctag gagcttgcgg ccgccagccc gggcaattcg aggggtatcg
     4321 ctcttgaagg gaactatgtt gaataataaa aacgaatttt gacaaaaaaa tgtgtttttc
     4381 tttgttagac cggggactta tcagccaacc tgttatgtgg cgcggtatta tcccgtattg
     4441 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4501 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4561 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcgaggcga
     4621 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa
     4681 ttaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     4741 taataatggt ttcttagagc ttacggccag cctcgcagag caggattccc gttgagcacc
     4801 gccaggtgcg aataagggac agtgaagaag gaacacccgc tcgcgggtgg gcctacttca
     4861 cctatcctgc ccggctgacg ccgttggata caccaaggaa agtctacacg aaccctttgg
     4921 caaaatcctg tatatcgtgc gaaaaaggat ggatataccg aaaaaatcgc tataatgacc
     4981 ccgaagcagg gttatgcagc gggtgagcgc aacgcaatta atgtgagtta gctcactcat
     5041 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     5101 ggataacaat ttcacacagg aaacacatat ggaaaaaaag gaatttcgtg ttttgataaa
     5161 atactgtttt ctgaagggaa aaaatacggt ggaagcaaaa acttggcttg ataatgagtt
     5221 tccggactct gccccaggga aatcaacaat aattgattgg tatgcaaaat tcaagcgtgg
     5281 tgaaatgagc acggaggacg gtgaacgcag tggacgcccg aaagaggtgg ttaccgacga
     5341 aaacatcaaa aaaatccaca aaatgatttt gaatgaccgt aaaatgaagt tgatcgagat
     5401 agcagaggcc ttaaagatat caaaggaacg tgttggtcat atcattcatc aatatttgga
     5461 tatgcggaag ctctgtgcaa aatgggtgcc gcgcgagctc acatttgacc aaaaacaaca
     5521 acgtgttgat gattctgagc ggtgtttgca gctgttaact cgtaatacac ccgagttttt
     5581 ccgtcgatat gtgacaatgg atgaaacatg gctccatcac tacactcctg agtccaatcg
     5641 acagtcggct gagtggacag cgaccggtga accgtctccg aagcgtggaa agactcaaaa
     5701 gtccgctggc aaagtaatgg cctctgtttt ttgggatgcg catggaataa tttttatcga
     5761 ttatcttgag aagggaaaaa ccatcaacag tgactattat atggcgttat tggagcgttt
     5821 gaaggtcgaa atcgcggcaa aacggcccca catgaagaag aaaaaagtgt tgttccacca
     5881 agacaacgca ccgtgccaca agtcattgag aacgatggca aaaattcatg aattgggctt
     5941 cgaattgctt ccccacccac cgtattctcc agatctggcc cccagcgact ttttcttgtt
     6001 ctcagacctc aaaaggatgc tcgcagggaa aaaatttggc tgcaatgaag aggtgatcgc
     6061 cgaaactgag gcctattttg aggcaaaacc gaaggagtac taccaaaatg gtatcaaaaa
     6121 attggaaggt cgttataatc gttgtatcgc tcttgaaggg aactatgttg aataataaaa
     6181 acgaattttg acaaaaaaat gtgtttttct ttgttagacc ggaatctgca gaattccagc
     6241 acactggcgg ccgtta
//
