LOCUS       Exported                5089 bp DNA     circular SYN 10-AUG-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 5089)
  AUTHORS   ASTRID HAASE
  TITLE     Direct Submission
  JOURNAL   Exported May 20, 2022 from SnapGene Viewer 6.0.5
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5089
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     protein_bind    505..526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        541..571
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..595
                     /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     603..619
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     613..632
                     /label=CMA128
                     /note="Tm = 60.4   used to insert N-term tag into PuC19. 
                     Has homology to upstream HINDIII cut site in pUC19 vector, 
                     homology  arm also includes BbSI cute site (sequence: 
                     GAAGAC)"
     primer_bind     613..632
                     /label=CMA141
                     /note="Tm = 62.5"
     primer_bind     613..632
                     /label=CMA136
                     /note="Tm = 60.4     End of primer has  homology to the 
                     lacZa sequence in pUC19, contains BbSI recognition site and
                      add 9 bps (AGAAACACC) because BbSI is a distance cutter 
                     (very similar to  CMA128 - just has added sequence to 
                     account for BbSI being a distance cutter)"
     CDS             615..650
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACR"
     primer_bind     631..670
                     /label=MQC70
     misc_feature    632..650
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1020..1038)
                     /label=MQC99
     primer_bind     1020..1038
                     /label=MQC98
     primer_bind     1146..1164
                     /label=MQC100
     primer_bind     complement(1146..1164)
                     /label=MQC101
     primer_bind     complement(1181..1221)
                     /label=MQC71
     misc_feature    1194..1201
                     /label=reverse sgRNA
     primer_bind     1202..1224
                     /label=CMA136
                     /note="Tm = 60.4     End of primer has  homology to the 
                     lacZa sequence in pUC19, contains BbSI recognition site and
                      add 9 bps (AGAAACACC) because BbSI is a distance cutter 
                     (very similar to  CMA128 - just has added sequence to 
                     account for BbSI being a distance cutter)"
     primer_bind     1203..1229
                     /label=SS197
     primer_bind     1203..1224
                     /label=CMA128
                     /note="Tm = 60.4   used to insert N-term tag into PuC19. 
                     Has homology to upstream HINDIII cut site in pUC19 vector, 
                     homology  arm also includes BbSI cute site (sequence: 
                     GAAGAC)"
     primer_bind     1204..1229
                     /label=SS202
     primer_bind     1204..1229
                     /label=SS199
     primer_bind     1204..1227
                     /label=CMA101
     primer_bind     1204..1223
                     /label=AE37
     primer_bind     1204..1223
                     /label=AE42
     misc_feature    1204..1212
                     /label=splice donor
     misc_feature    1204..1212
                     /label=THIS WAS LEFT OUT ON ACCIDENT IN 6.25.18
     primer_bind     1211..1237
                     /label=SS204
                     /note="61.8"
     polyA_signal    1213..1347
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1213..1237
                     /label=SS037
     primer_bind     1291..1310
                     /label=SV40pA-R
     primer_bind     1318..1335
                     /label=TK07
     CDS             complement(1348..1944)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1360..1374)
                     /label=DS4
     primer_bind     complement(1434..1450)
                     /label=DS7
     primer_bind     1794..1812
                     /label=SS186
     misc_feature    1895..1896
                     
                     /note="Clone 1 has a C inserted between these two bps "
     primer_bind     complement(1932..1947)
                     /label=TK03
     misc_feature    1943..1944
                     
                     /note="Clone 1 has a G inserted between these two base 
                     pairs "
     misc_feature    1945..1947
                     /label=start
     misc_feature    1948..1954
                     /label=kozak
     misc_feature    1955..2254
                     /label=act5c
     primer_bind     1955..1975
                     /label=CMA140
                     /note="Tm = 62.1"
     primer_bind     complement(1955..1973)
                     /label=TK06
     primer_bind     complement(2110..2125)
                     /label=TK08
     primer_bind     complement(2231..2254)
                     /label=CMA141
                     /note="Tm = 62.5"
     primer_bind     2255..2283
                     /label=MQC-56
     misc_feature    2255..2259
                     /label=branch
     misc_feature    2261..2265
                     /label=branch
     misc_feature    2276..2291
                     /label=Y(11)N(C/T)AG|G
     misc_feature    2276
                     /label=A>C
     misc_feature    2277..2286
                     /label=pyrimidine tract
     misc_feature    2287
                     /label=N
     misc_feature    2288
                     /label=c/t
     misc_feature    2289..2290
                     /label=AG
     misc_feature    2291
                     /label=C>G
     misc_feature    2292..2298
                     /label=Kozak
     misc_feature    2299..2301
                     /label=Start
     CDS             2302..2367
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     2302..2326
                     /label=AE26
     primer_bind     2302..2321
                     /label=AE45
     misc_feature    2368..2403
                     /label=ggs linker
                     /label=Linker(1)
                     /note="ApEinfo_fwdcolor=#d0cbcf
                     ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} 
                     width 5 offset 0
                     ApEinfo_label=Linker
                     ApEinfo_revcolor=#d0cbcf"
     CDS             2404..2493
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     primer_bind     complement(2462..2485)
                     /label=SS205
                     /note="62"
     primer_bind     complement(2470..2520)
                     /label=SS201
     primer_bind     complement(2470..2493)
                     /label=SS203
     primer_bind     complement(2474..2493)
                     /label=AE38
     misc_feature    2494..2520
                     /label=ggs linker. forgot in 1st design.
     primer_bind     2501..2539
                     /label=mqc72
     primer_bind     complement(2501..2520)
                     /label=AE43
                     /note="Primer used to PCR out N-term tag"
     primer_bind     2501..2520
                     /label=MQC-22
     primer_bind     complement(2503..2520)
                     /label=CMA102
     primer_bind     complement(2505..2520)
                     /label=CMA129
                     /note="Tm = 60.2˚      used to insert N-term tag into 
                     PuC19. Has  homology to downstream HINDIII cut site in 
                     pUC19 vector, homology arm also  includes BbSI cute site 
                     (sequence: GAAGAC)"
     primer_bind     complement(2505..2520)
                     /label=CMA137
                     /note="Tm = 60.2           End of primer has homology to 
                     the lacZa  sequence in pUC19, contains BbSI recognition 
                     site and add 9 bps (ATAATAATA)  because BbSI is a distance 
                     cutter (very similar to CMA129 - just has added  sequence 
                     to account for BbSI being a distance cutter)"
     misc_feature    2521..2573
                     /label=start
     misc_feature    2521..2532
                     /label=reverse sgRNA
     primer_bind     complement(3043..3083)
                     /label=mqc73
     CDS             3070..3341
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="RVPSSNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDAA"
     misc_feature    3070..3091
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3092..3108)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3582..3686
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3687..4547
                     /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"
     rep_origin      join(4718..5089,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      541 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      601 cacaggaaac agctatgacc atgattacgc caagcttgca tgcctgcagg ggtctacgga
      661 gctgagtatc tttgtgagaa aactaccgaa gaagaaggca gttatggctg tgaagaaagt
      721 cactaaatta tttaatcgcc tcatttatgt gttgttttga ctagagagac cgtgaaacat
      781 tcgcgaaaat ataccatgtt tactggggta tcgcaaattt gttcattgaa tctcttgctt
      841 gcattgaatg aataatcgtt aattttggtt ttatcataaa gtggaaagca aattatatag
      901 actgtatact tatgtatact gtcttaaaag taagttaaat aaagtttgca ctaataatat
      961 attttaaaaa ttcgagttcg gaatcgctgg accaaagcat ttcaattcgt aagtaagacc
     1021 gtatcccctt aacaataacg gaatcctgat tgccgcccaa tgcgagtaat attaaattga
     1081 gttggcagtc ttatgctata tttttgttga ttaaaaagtg taaaaacata ttcgaaatta
     1141 aaagtagtaa tcgtattaag acactgggtt gtgattggcg ctattcgaga ccttttagag
     1201 ccccaggtaa gtgatccaga catgataaga tacattgatg agtttggaca aaccacaact
     1261 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     1321 accattataa gctgcaataa acaagttcta ggcaccgggc ttgcgggtca tgcaccaggt
     1381 gcgcggtcct tcgggcacct cgacgtcggc ggtgacggtg aagccgagcc gctcgtagaa
     1441 ggggaggttg cggggcgcgg aggtctccag gaaggcgggc accccggcgc gctcggccgc
     1501 ctccactccg gggagcacga cggcgctgcc cagacccttg ccctggtggt cgggcgagac
     1561 gccgacggtg gccaggaacc acgcgggctc cttgggccgg tgcggcgcca ggaggccttc
     1621 catctgttgc tgcgcggcca gccgggaacc gctcaactcg gccatgcgcg ggccgatctc
     1681 ggcgaacacc gcccccgctt cgacgctctc cggcgtggtc cagaccgcca ccgcggcgcc
     1741 gtcgtccgcg acccacacct tgccgatgtc gagcccgacg cgcgtgagga agagttcttg
     1801 cagctcggtg acccgctcga tgtggcggtc cgggtcgacg gtgtggcgcg tggcggggta
     1861 gtcggcgaac gcggcggcga gggtgcgtac ggcccggggg acgtcgtcgc gggtggcgag
     1921 gcgcaccgtg ggcttgtact cggtcatttt gattgtctct ggattagacg actgctggct
     1981 gatggagcgg ctttgtgtcg ggaggagtat ccacacagca caagaactca aacggtagtg
     2041 atatgaaaac tggtcccgca gcccgtttta taaagcccgc ttagcgctaa aaccttatca
     2101 gccgcaatgc tctccgctct caagtcgcgt tcaaaacttt taccatatat gggtatgaat
     2161 tgttcgccga ttgggttttc atttacggag aatttcctcc gcaactgggt gttcgtgctt
     2221 gtgtgtgtct tgtatatcgc catgaagagt gtacctaatt ttaatcaacc gatttctctc
     2281 tcttccgcag gaatcaaaat ggactacaaa gaccatgacg gtgattataa agatcatgac
     2341 atcgattaca aggatgacga tgacaagggt ggatctggag gttccggcgg ctcagggggt
     2401 agttacccat acgatgttcc tgactatgcg ggctatccct atgacgtccc ggactatgca
     2461 ggatcctatc catatgacgt tccagattac gcttccggtg gctctggctc ttcaggctca
     2521 atggacgaca tgatgcgggc gatggactgt ctaaatatgg cagcgacgtt gcgcaacgcg
     2581 gctctggcgt ccgcaacgtg cacgggcagg actttgggcg aggatcggga acctatatgg
     2641 gtggagggca gtcggaagac acctgagcca ccattacccg aagaaagccc agctccagag
     2701 cccaataacg agattcccct gctgcccaaa atagattttg agcctaatat atcctgcttt
     2761 cccgacctac aggctatcaa cgctagcatt gttaggcggg aactggagga ggagggtaag
     2821 aggtgtgtcc gccagcagct aaaggcgata agggacaaac aggacgccat gcgcctgtcg
     2881 cgagagacgc agcaaaggaa ggaggaacgc cagcgcgacc agttgcaaca aaaggcgctg
     2941 cgggaacgca atgaaagtct ccttatccag aaagctgacc agatgactgc ggcacaacta
     3001 gaggcccagc agcgcgagca attggcactg cgccaacaga ttgaccagaa gctgcacaag
     3061 ttgaggatcc ccgggtaccg agctcgaatt cactggccgt cgttttacaa cgtcgtgact
     3121 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     3181 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     3241 gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3301 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     3361 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3421 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3481 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     3541 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3601 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3661 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3721 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3781 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3841 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3901 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     3961 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     4021 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     4081 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     4141 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     4201 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     4261 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     4321 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     4381 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     4441 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     4501 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4561 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4621 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4681 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4741 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4801 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4861 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4921 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4981 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5041 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaact
//
