LOCUS       Exported                5442 bp DNA     circular SYN 23-MAR-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5442)
  AUTHORS   EJPARKER
  TITLE     Direct Submission
  JOURNAL   Exported Apr 27, 2022 from SnapGene 6.0.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5442
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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.FOR
     primer_bind     1380..1405
                     /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     1390..1426
                     /label=FWD_primer_PnptII
     CDS             complement(1406)
                     /codon_start=1
                     /label='cat
                     /translation=""
     promoter        1417..1753
                     /label=p-nptII promoter
                     /label=p-nptII
     primer_bind     1417..1436
                     /label=11x-nptII-FL-fw
     primer_bind     1417..1431
                     /label=lederm_nptII.fw
     primer_bind     1567..1585
                     /label=pFru97xpMRE100_nptIIGib_rv
     primer_bind     complement(1752..1784)
                     /label=REV_primer_PnptII_mTq2/sG2
     primer_bind     complement(1752..1781)
                     /label=REV_primer_PnptII_mCl/mO/mSc/sY2
     primer_bind     complement(1752..1777)
                     /label=REV_primer_PnptII_mTagBFP2
     primer_bind     1762..1794
                     /label=FWD_primer_mTq2/sGFP2
     primer_bind     1762..1791
                     /label=FWD_primer_mCl/mO/mSc/sY2
     primer_bind     1770..1787
                     /label=XhoI_Ptac_XFP_for
     CDS             1772..2488
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     1772..1791
                     /label=FWD_sYFP2
     primer_bind     1772..1789
                     /label=XFPs for
     primer_bind     complement(1817..1838)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2293..2313)
                     /label=FWD_primer_PnptII
     primer_bind     2425..2446
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2439..2458)
                     /label=REV_primer_sG2
     primer_bind     complement(2469..2511)
                     /label=REV_primer_mTq2
     primer_bind     complement(2470..2493)
                     /label=NotI_XFP_rev
     primer_bind     complement(2470..2491)
                     /label=nextgenFP.rv
     primer_bind     complement(2472..2511)
                     /label=REV_primer_mO
     primer_bind     complement(2472..2491)
                     /label=11x-FL-rv
     primer_bind     complement(2473..2511)
                     /label=REV_primer_sY2
     primer_bind     complement(2473..2491)
                     /label=REV_sYFP2
     primer_bind     complement(2473..2491)
                     /label=Ptac+eXFP_rev
     primer_bind     complement(2474..2491)
                     /label=XFPs rev KpnI
     primer_bind     2492..2506
                     /label=Gib_Gent.rev
     primer_bind     complement(2499..2518)
                     /label=FWD_pFru_ins
     primer_bind     2502..2524
                     /label=Gib_Gent.for.cor
     terminator      2508..2602
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2525..2543)
                     /label=pQE-R
                     /note="19"
     CDS             2646..3305
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2707..2735)
                     /label=pMRE130inserts_rev
     primer_bind     complement(3290..3305)
                     /label=Chloramp_rev
     terminator      3370..3456
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3461..3478)
                     /label=Gib_Gent.for
     primer_bind     complement(3466..3505)
                     /label=Fragment.REV
     primer_bind     complement(3466..3483)
                     /label=pMRE130_cassette2_RV
     primer_bind     complement(3467..3483)
                     /label=cassette_pAG408overhang_RV
     primer_bind     3484..3510
                     /label=pHimarEm1+XmaI_FWD
     repeat_region   3573..3601
                     /label=IR2
     primer_bind     3847..3863
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3943..3954)
                     /label=cassette_pAG408overhang_RV
     oriT            3950..4058
                     /note="incP origin of transfer"
     protein_bind    4205..4226
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4241..4271
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     promoter        4241..4270
                     /gene="lac prom"
                     /label=lac prom promoter
     protein_bind    4279..4295
                     /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    4315..5361
                     /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 atcggattag agctcgcacg ctgccgcaag cactcagggc
     1441 gcaagggctg ctaaaggaag cggaacacgt agaaagccag tccgcagaaa cggtgctgac
     1501 cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc gcaaagagaa
     1561 agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt ttatggacag
     1621 caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag
     1681 taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca agatctgatc
     1741 aagagacagg atactagtgg aggaagaaaa aatggtgagc aagggcgagg agctgttcac
     1801 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1861 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1921 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1981 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     2041 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     2101 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     2161 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     2221 cgtctatatc accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca
     2281 caacatcgag gacggcggcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     2341 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtcca agctgagcaa
     2401 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2461 tacacatggc atggacgagc tgtacaagta ataattagct gagcttggac tcctgttgat
     2521 agatccagta atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg
     2581 ggcgtttttt attggtgaga atccaagcta gcttggcgag attttcagga gctaaggaag
     2641 ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta
     2701 aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc
     2761 tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct
     2821 ttattcacat tcttgcccgc ctgatgaatg ctcatccgga atttcgtatg gcaatgaaag
     2881 acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa
     2941 ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca
     3001 tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta
     3061 ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa
     3121 acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc
     3181 aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtc tgtgatggct
     3241 tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg
     3301 cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc tggggtaatg actctctagc
     3361 ttgaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     3421 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgctaggagc ttgcggccgc
     3481 cagcccgggc aattcgaggg gtatcgctct tgaagggaac tatgttgaat aataaaaacg
     3541 aattttgaca aaaaaatgtg tttttctttg ttagaccggg gacttatcag ccaacctgtt
     3601 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     3661 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3721 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3781 cttacttctg acaacgatcg aggcgattaa gttgggtaac gccagggttt tcccagtcac
     3841 gacgttgtaa aacgacggcc agtgaattaa ttcttgaaga cgaaagggcc tcgtgatacg
     3901 cctattttta taggttaatg tcatgataat aatggtttct tagagcttac ggccagcctc
     3961 gcagagcagg attcccgttg agcaccgcca ggtgcgaata agggacagtg aagaaggaac
     4021 acccgctcgc gggtgggcct acttcaccta tcctgcccgg ctgacgccgt tggatacacc
     4081 aaggaaagtc tacacgaacc ctttggcaaa atcctgtata tcgtgcgaaa aaggatggat
     4141 ataccgaaaa aatcgctata atgaccccga agcagggtta tgcagcgggt gagcgcaacg
     4201 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     4261 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac acatatggaa
     4321 aaaaaggaat ttcgtgtttt gataaaatac tgttttctga agggaaaaaa tacggtggaa
     4381 gcaaaaactt ggcttgataa tgagtttccg gactctgccc cagggaaatc aacaataatt
     4441 gattggtatg caaaattcaa gcgtggtgaa atgagcacgg aggacggtga acgcagtgga
     4501 cgcccgaaag aggtggttac cgacgaaaac atcaaaaaaa tccacaaaat gattttgaat
     4561 gaccgtaaaa tgaagttgat cgagatagca gaggccttaa agatatcaaa ggaacgtgtt
     4621 ggtcatatca ttcatcaata tttggatatg cggaagctct gtgcaaaatg ggtgccgcgc
     4681 gagctcacat ttgaccaaaa acaacaacgt gttgatgatt ctgagcggtg tttgcagctg
     4741 ttaactcgta atacacccga gtttttccgt cgatatgtga caatggatga aacatggctc
     4801 catcactaca ctcctgagtc caatcgacag tcggctgagt ggacagcgac cggtgaaccg
     4861 tctccgaagc gtggaaagac tcaaaagtcc gctggcaaag taatggcctc tgttttttgg
     4921 gatgcgcatg gaataatttt tatcgattat cttgagaagg gaaaaaccat caacagtgac
     4981 tattatatgg cgttattgga gcgtttgaag gtcgaaatcg cggcaaaacg gccccacatg
     5041 aagaagaaaa aagtgttgtt ccaccaagac aacgcaccgt gccacaagtc attgagaacg
     5101 atggcaaaaa ttcatgaatt gggcttcgaa ttgcttcccc acccaccgta ttctccagat
     5161 ctggccccca gcgacttttt cttgttctca gacctcaaaa ggatgctcgc agggaaaaaa
     5221 tttggctgca atgaagaggt gatcgccgaa actgaggcct attttgaggc aaaaccgaag
     5281 gagtactacc aaaatggtat caaaaaattg gaaggtcgtt ataatcgttg tatcgctctt
     5341 gaagggaact atgttgaata ataaaaacga attttgacaa aaaaatgtgt ttttctttgt
     5401 tagaccggaa tctgcagaat tccagcacac tggcggccgt ta
//
