LOCUS       Type2B_CombiCR_s        4724 bp DNA     circular SYN 08-JUN-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4724)
  AUTHORS   MIT
  TITLE     Direct Submission
  JOURNAL   Exported Dec 12, 2023 from SnapGene 7.1.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4724
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..4
                     /label=BsaI
     primer_bind     6..38
                     /label=MAA_o512
     misc_feature    19..43
                     /label=ConS scar
     protein_bind    19..22
                     /label=BsmBI
     misc_feature    25..27
                     /label=PAM
     misc_feature    50..58
                     /label=Add BglII site
     misc_feature    59..93
                     /label=Optional spacer / connector
     protein_bind    59..62
                     /label=BsaI
     misc_feature    63..87
                     /label=Con1 scar
     protein_bind    63..66
                     /label=BsmBI
     primer_bind     complement(80..97)
                     /label=MAA_o531
     protein_bind    88..91
                     /label=BsaI
     protein_bind    94..97
                     /label=BsaI
     primer_bind     97..124
                     /label=MAA_o340
     misc_feature    98..682
                     /label=order_gblock
     misc_feature    98..205
                     /label=Ptet_voigt_v3
     primer_bind     98..133
                     /label=MAA_o530
     primer_bind     98..122
                     /label=oENG491_ytk
     misc_feature    98..118
                     /label=nucleosome depletion region
     misc_feature    123..130
                     /label=TATA box
     protein_bind    131..149
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     138..197
                     /label=MAA_o254
     primer_bind     138..197
                     /label=MAA_o258
     primer_bind     138..197
                     /label=MAA_o276
     misc_feature    150..169
                     /label=T20-spacer
     primer_bind     complement(168..206)
                     /label=MAA_o373
     protein_bind    170..188
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(177..205)
                     /label=oENG492_ytk
     primer_bind     complement(179..211)
                     /label=MAA_o263
     primer_bind     complement(180..211)
                     /label=MAA_o341
     misc_feature    186..205
                     /label=TSS
     primer_bind     208..243
                     /label=MAA_o281
     CDS             209..277
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="MDYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    278..328
                     /label=conserved
     primer_bind     278..307
                     /label=MAA_o286
     CDS             278..283
                     /codon_start=1
                     /label=start
                     /translation="MA"
     CDS             278..280
                     /codon_start=1
                     /label=added_via_ytk
                     /translation="M"
     CDS             284..304
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             329..337
                     /gene="GSGS-PDZ"
                     /label=GSG
                     /note="/SECDrawAs=Gene"
     CDS             344..346
                     /gene="43-8-WT-RIGHT"
                     /label=43-8-WT-RIGHT
                     /note="/SECDrawAs=Gene"
     CDS             347..601
                     /codon_start=1
                     /label=sTF_42-10_HA_opt
                     /translation="GERPFQCRICMANFSTGQILDRHTRTHTGEKPFQCRICMANFSVA
                     HSLKRHLRTHTGEKPFQCRICMANFSDPSNLRRHLKTHLR"
     primer_bind     347..366
                     /label=oENG325_ytk
     CDS             353..355
                     /codon_start=1
                     /label=changed for BsaI site
                     /translation="R"
     CDS             392..412
                     /codon_start=1
                     /label=F1
                     /translation="TGQILDR"
     misc_feature    434..447
                     /label=homologous with 97-4
     misc_feature    439
                     /label=changed from G to avoid homology
     CDS             476..496
                     /codon_start=1
                     /label=F2
                     /translation="VAHSLKR"
     CDS             560..580
                     /codon_start=1
                     /label=F3
                     /translation="DPSNLRR"
     primer_bind     complement(568..603)
                     /label=oENG326_ytk
     primer_bind     complement(586..606)
                     /label=oENG467_ytk
     misc_feature    608..658
                     /label=17aa GS linker
     misc_feature    660..667
                     /label=SbfI
     misc_feature    671..673
                     /label=changed to tct
     primer_bind     683..701
                     /label=MAA_o391
     primer_bind     683..701
                     /label=MAA_o408
     misc_feature    683..685
                     /label=stop_to_insert
     terminator      686..909
                     /label=ScTDH1 Terminator
     primer_bind     686..701
                     /label=MAA_o383
     misc_feature    910..917
                     /label=connection_scar
     misc_feature    918..969
                     /label=ribozyme
     misc_feature    970..1067
                     /label=spacer
     primer_bind     complement(1048..1101)
                     /label=MAA_o409
     primer_bind     complement(1056..1069)
                     /label=MAA_o384
     protein_bind    1070..1073
                     /label=BsaI
     primer_bind     complement(1100..1123)
                     /label=MAA_o375
     protein_bind    1126..1129
                     /label=BsaI
     misc_feature    1130..1154
                     /label=ConE scar
     primer_bind     1130..1152
                     /label=MAA_o205
     protein_bind    1130..1133
                     /label=BsmBI
     protein_bind    1170..1173
                     /label=BsaI
     promoter        1174..1410
                     /label=HIS3 Promoter
     primer_bind     complement(1174..1193)
                     /label=MAA_o202
     primer_bind     complement(1389..1413)
                     /label=org351_his3_tag_2
     CDS             1411..2073
                     /label=HIS3
     primer_bind     1414..1437
                     /label=org350_his3_tag_1
     primer_bind     1414..1437
                     /label=org371_his3_ttrz_f
     primer_bind     1670..1688
                     /label=MAA_o206
     primer_bind     complement(2049..2073)
                     /label=org367_his3_r_bamhi
     terminator      2074..2283
                     /label=HIS3 Terminator
     primer_bind     2273..2292
                     /label=MAA_o207
     protein_bind    2284..2287
                     /label=BsaI
     rep_origin      2288..2404
                     /label=CEN6
     rep_origin      2405..2795
                     /label=ARS4
     protein_bind    2796..2799
                     /label=BsaI
     CDS             complement(2808..3818)
                     /label=SpecR
     CDS             complement(2808..3599)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     primer_bind     2808..2827
                     /label=SpecR_pOP_r
     promoter        complement(3819..3952)
                     /label=SpecR Promoter
     rep_origin      complement(3953..4716)
                     /direction=LEFT
                     /label=ColE1
     primer_bind     complement(3954..3974)
                     /label=SpecR_pOP_f
     rep_origin      4029..4617
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ccctgaatta catctagact gaactggccg ataattgcag acgaacgctg aagatctagc
       61 tgccaaacca gatgtcaaca cagctacaac gctgctgttt ttttattttt tgttttttgc
      121 ggtataaaaa tccctatcag tgatagagat tttatttttt ttttcttttt ccctatcagt
      181 gatagagaat atcaagctac aaaaaggtat ggattataaa gaccatgacg gagattataa
      241 agatcatgac atagattaca aggatgacga tgacaagatg gcaccaaaga agaagaggaa
      301 ggtgggtatt cacggagtcc ccggaggtgg ctcagggtcc cgtcccggtg agaggccctt
      361 tcaatgcaga atttgcatgg caaacttttc cacggggcaa atcttggatc gtcatacaag
      421 aacgcatacg ggcgaaaaac cattccaatg ccgtatttgc atggctaact ttagcgttgc
      481 gcactccttg aagaggcacc tgagaacaca tactggcgag aagccctttc agtgcagaat
      541 ctgtatggcg aatttctccg atccctccaa tcttcgtaga catttgaaaa ctcatttaag
      601 aggatctggc ggtagtggcg gagggagtgg tggcgggggc tctggaggcg gatctgggtc
      661 ctgcaggggt tctgtcttct aataaataaa gcaatcttga tgaggataat gatttttttt
      721 tgaatataca taaatactac cgtttttctg ctagattttg tgatgacgta aataagtaca
      781 tattactttt taagccaaga caagattaag cattaacttt acccttttct ttctaagttt
      841 caatattagt tatcactgtt taaaagttat ggcgagaacg tcggcggtta aaatatatta
      901 ccctgaacgc ctcttccagc gctcaacggg tgtgcttccc gttctgatga gtccgtgagg
      961 acgaaagcgt cccgggaata tcgcgtagca ctttagactc ttccttacgt aatggatgac
     1021 tcggcgcgtg atcttacggc cgggcgacac ctctcatagg tgtgcgtgga tcctaacttt
     1081 tttggatccg caacggaatt cctgacgcac aattgttgtt gtttggctga gcagttacag
     1141 agatgttacg aaccactagt gcactgcagt acaaacacag tcctttcccg caattttctt
     1201 tttctattac tcttggcctc ctctagtaca ctctatattt ttttatgcct cggtaatgat
     1261 tttcattttt ttttttccac ctagcggatg actctttttt tttcttagcg attggcatta
     1321 tcacataatg aattatacat tatataaagt aatgtgattt cttcgaagaa tatactaaaa
     1381 aatgagcagg caagataaac gaaggcaaag atgacagagc agaaagccct agtaaagcgt
     1441 attacaaatg aaaccaagat tcagattgcg atctctttaa agggtggtcc cctagcgata
     1501 gagcactcga tcttcccaga aaaagaggca gaagcagtag cagaacaggc cacacaatcg
     1561 caagtgatta acgtccacac aggtataggg tttctggacc atatgataca tgctctggcc
     1621 aagcattccg gctggtcgct aatcgttgag tgcattggtg acttacacat agacgaccat
     1681 cacaccactg aggactgcgg gattgctctc ggtcaagctt ttaaagaggc cctaggggcc
     1741 gtgcgtggag taaaaaggtt tggatcagga tttgcgcctt tggatgaggc actttccaga
     1801 gcggtggttg atctttcgaa caggccgtac gcagttgtcg aacttggttt gcaaagggag
     1861 aaagtaggtg atctctcttg cgagatgatc ccgcattttc ttgaaagctt tgcagaggct
     1921 agcagaatta ccctccacgt tgattgtctg cgaggcaaga atgatcatca ccgtagtgag
     1981 agtgcgttca aggctcttgc ggttgccata agagaagcca cctcgcccaa tggtaccaac
     2041 gatgttccct ccaccaaagg tgttcttatg tagtgacacc gattatttaa agctgctgca
     2101 tacgatatat atacatgtgt atatatgtat acctatgaat gtcagtaagt atgtatacga
     2161 acagtatgat actgaagatg acaaggtaat gcatcattct atacgtgtca ttctgaacga
     2221 ggcgcgcttt ccttttttct ttttgctttt tctttttttt tctcttgaac tcgacggatc
     2281 atagagtatc acgtgctata aaaataatta taatttaaat tttttaatat aaatatataa
     2341 attaaaaata gaaagtaaaa aaagaaatta aagaaaaaat agtttttgtt ttccgaagat
     2401 gtaaaagact ctagggggat cgccaacaaa tactaccttt tatcttgctc ttcctgctct
     2461 caggtattaa tgccgaattg tttcatcttg tctgtgtaga aaaccacaca cgaaaatcct
     2521 gtgattttac attttactta tcgttaatcg aatgtatatc tatttaatct gcttttcttg
     2581 tctaataaat atatatgtaa agtacgcttt ttgttgaaat tttttaaacc tttgtttatt
     2641 tttttttctt cattccgtaa ctcttctacc ttctttattt actttctaaa atccaaatac
     2701 aaaacataaa aataaataaa cacagagtaa attcccaaat tattccatca ttaaaagata
     2761 cgaggcgcgt gtaagttaca ggcaagcgat ccgtcccgag cggccgctta tttgccgact
     2821 accttggtga tctcgccttt cacgtagtgg acaaattctt ccaactgatc tgcgcgcgag
     2881 gccaagcgat cttcttcttg tccaagataa gcctgtctag cttcaagtat gacgggctga
     2941 tactgggccg gcaggcgctc cattgcccag tcggcagcga catccttcgg cgcgattttg
     3001 ccggttactg cgctgtacca aatgcgggac aacgtaagca ctacatttcg ctcatcgcca
     3061 gcccagtcgg gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc aaatagatcc
     3121 tgttcaggaa ccggatcaaa gagttcctcc gccgctggac ctaccaaggc aacgctatgt
     3181 tctcttgctt ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg ctcgaagata
     3241 cccgcaagaa tgtcattgcg ctgccattct ccaaattgca gttcgcgctt agctggataa
     3301 cgccacggaa tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg gagaatctcg
     3361 ctctctccag gggaagccga agtttccaaa aggtcgttga tcaaagctcg ccgcgttgtt
     3421 tcatcaagcc ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg cttcaggccg
     3481 ccatccactg cggagccgta caaatgtacg gccagcaacg tcggttcgag atggcgctcg
     3541 atgacgccaa ctacctctga tagttgagtc gatacttcgg cgatcaccgc ttccctcata
     3601 atgtttaact ttgttttagg gcgactgccc tgctgcgtaa catcgttgct gctccataac
     3661 atcaaacatc gacccacggc gtaacgcgct tgctgcttgg atgcccgagg catagactgt
     3721 accccaaaaa aacagtcata acaagccatg aaaaccgcca ctgcgccgtt accaccgctg
     3781 cgttcggtca aggttctgga ccagttgcgt gagcgcatac gctacttgca ttacagctta
     3841 cgaaccgaac aggcttatgt ccactgggtt cgtgccttca tccgtttcca cggtgtgcgt
     3901 cacccggcaa ccttgggtag cagcgaagtc gaggcatttc tgtcctggct ggtcatgacc
     3961 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4021 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4081 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4141 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4201 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4261 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4321 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4381 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4441 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4501 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4561 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4621 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4681 tttcctgcgt tatcccctga ttctgtggat aaccgtgcgg ccgc
//
