LOCUS       Exported                5410 bp ds-DNA     circular SYN 27-OCT-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5410)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Oct 27, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     Imported using the Genbank importer. File name: FRP2350.gb 
            finalP_tagging_casette This file was created using Genome Compiler.
FEATURES             Location/Qualifiers
     source          1..5410
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     415..431
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        441..459
                     /label=T7_promoter Promoter|promoter
     primer_bind     441..459
                     /label=FRO4535
     primer_bind     483..502
                     /label=FRO4549
     primer_bind     483..502
                     /label=FRO4477
     misc_feature    484..862
                     /label=P_TEF1
     primer_bind     484..502
                     /label=FRO4479
     primer_bind     484..502
                     /label=FRO4454
     primer_bind     484..502
                     /label=FRO4531
     primer_bind     484..502
                     /label=FRO4552
     primer_bind     484..502
                     /label=FRO4510
     primer_bind     484..502
                     /label=FRO4474
     primer_bind     514..533
                     /label=FRO351
     gene            519..2094
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        519..862
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     833..862
                     /label=FRO4538
     primer_bind     complement(833..862)
                     /label=FRO4540
     CDS             863..1891
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     primer_bind     863..885
                     /label=FRO4627
     misc_feature    1892..2128
                     /label=term_TEF1
     primer_bind     1892..1921
                     /label=FRO4541
     primer_bind     complement(1892..1921)
                     /label=FRO4539
     terminator      1897..2094
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2119..2143)
                     /label=FRO4451
     primer_bind     complement(2119..2143)
                     /label=FRO4480
     primer_bind     2124..2178
                     /label=o6042
     primer_bind     complement(2127..2150)
                     /label=FRO4299
     primer_bind     2130..2174
                     /label=FRO4300
     misc_feature    2151..2875
                     /label=P7Sul.1
     primer_bind     2151..2178
                     /label=FRO4507
     primer_bind     complement(2156..2177)
                     /label=FRO4368
     primer_bind     complement(2163..2182)
                     /label=FRO4445
     primer_bind     complement(2229..2274)
                     /label=o5259
     misc_feature    2249..2264
                     /label=minSulA
     primer_bind     2255..2311
                     /label=o5260
     misc_feature    2264..2275
                     /label=RAP1binding
     misc_feature    2276..2291
                     /label=minSulA
     misc_feature    2291..2301
                     /label=GCR1binding
     primer_bind     2312..2331
                     /label=O5278
     misc_feature    2313..2322
                     /label=grf2_binding
     primer_bind     complement(2338..2396)
                     /label=FRO4981
     primer_bind     complement(2338..2356)
                     /label=FRO5013
     misc_feature    2343..2353
                     /label=grf2_binding
     misc_feature    2357..2372
                     /label=minSulA
     misc_feature    2372..2383
                     /label=RAP1binding
     misc_feature    2384..2399
                     /label=minSulA
     primer_bind     2397..2462
                     /label=FRO4980
     primer_bind     2399..2418
                     /label=FRO4293
     misc_feature    2399..2409
                     /label=GCR1binding
     primer_bind     complement(2410..2447)
                     /label=FRO5169
     misc_feature    2422..2437
                     /label=minSulA
     misc_feature    2437..2447
                     /label=GCR1binding
     primer_bind     2446..2475
                     /label=FRO5023
     primer_bind     2446..2462
                     /label=FRO5014
     primer_bind     2660..2683
                     /label=o5203
     primer_bind     complement(2660..2683)
                     /label=o5202
     primer_bind     complement(2710..2753)
                     /label=o5261
     misc_feature    2725..2745
                     /label=sulA
     misc_feature    2730..2747
                     /label=sulA.18
     misc_feature    2730..2745
                     /label=minSulA
     primer_bind     2733..2792
                     /label=o5262
     misc_feature    2746..2753
                     /label=TATAbox
     misc_feature    2754..2772
                     /label=sulA
     misc_feature    2754..2771
                     /label=sulA.18
     misc_feature    2754..2769
                     /label=minSulA
     primer_bind     2770..2790
                     /label=FRO4729
     primer_bind     complement(2837..2865)
                     /label=FRO5163
     primer_bind     2837..2855
                     /label=FRO0923
     primer_bind     2855..2883
                     /label=FRO4431
     primer_bind     complement(2855..2880)
                     /label=FRO5079
     misc_feature    2866..2880
                     /label=good kozak
     gap             2884..2955
                     /label=FLAG TAG|Unknown
     CDS             2884..2955
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     primer_bind     2930..3019
                     /label=FRO4298
     gap             2956..2979
                     /label=8G linker
     primer_bind     complement(2980..3002)
                     /label=FRO4532
     primer_bind     2980..3001
                     /label=o5477
     primer_bind     2980..2999
                     /label=S4 primer site
     primer_bind     complement(2980..2999)
                     /label=FRO4478
     primer_bind     complement(3000..3024)
                     /label=FRO4432
     promoter        complement(3017..3036)
                     /label=T3_promoter
     primer_bind     complement(3017..3036)
                     /label=FRO0114
     promoter        complement(3018..3036)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3053..3078)
                     /label=O5619
     primer_bind     complement(3057..3073)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3057..3073)
                     /label=M13r
     primer_bind     3071..3095
                     /label=FRO2169
     primer_bind     complement(3071..3090)
                     /label=LacOp-for
     primer_bind     complement(3079..3099)
                     /label=o5204
     protein_bind    3081..3097
                     /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)."
     promoter        complement(3105..3135)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3105..3124)
                     /label=pUCM13-rev-157
     protein_bind    3150..3171
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3187..3209)
                     /label=o5525
     primer_bind     3187..3206
                     /label=o5946
     rep_origin      complement(3459..4047)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4218..5078)
                     /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"
     promoter        complement(5079..5183)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5228..5410,1..273))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
       61 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
      121 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
      181 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
      241 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
      301 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
      361 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
      421 cgacggccag tgagcgcgcg taatacgact cactataggg cgaattggag ctcgatcctc
      481 gagagcttgc cttgtccccg ccgggtcacc cggccagcga catggaggcc cagaataccc
      541 tccttgacag tcttgacgtg cgcagctcag gggcatgatg tgactgtcgc ccgtacattt
      601 agcccataca tccccatgta taatcatttg catccataca ttttgatggc cgcacggcgc
      661 gaagcaaaaa ttacggctcc tcgctgcgga cctgcgagca gggaaacgct cccctcacag
      721 acgcgttgaa ttgtccccac gccgcgcccc tgtagagaaa tataaaaggt taggatttgc
      781 cactgaggtt cttctttcat atacttcctt ttaaaatctt gctaggatac agttctcaca
      841 tcacatccga acataaacaa ccatgggtaa aaagcctgaa ctcaccgcga cgtctgtcga
      901 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
      961 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     1021 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     1081 cccgattccg gaagtgcttg acattgggga gttcagcgag agcctgacct attgcatctc
     1141 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     1201 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     1261 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
     1321 cgcgattgct gatccccacg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     1381 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
     1441 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
     1501 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
     1561 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     1621 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     1681 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     1741 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     1801 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     1861 ccccagcact cgtccgaggg caaaggaata atcagtactg acaataaaaa gattcttgtt
     1921 ttcaagaact tgtcatttgt atagtttttt tatattgtag ttgttctatt ttaatcaaat
     1981 gttagcgtga tttatatttt ttttcgcctc gacatcatct gcccagatgc gaagttaagt
     2041 gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg
     2101 attcgatact aacgccgcca tccagtgttt aaacgaacta gtgcggccgc cagttcgagt
     2161 ttatcattat caatactgcc atttcaaaga atacgtaaat aattaatagt agtgattttc
     2221 ctaactttat ttagtcaaaa aattagccct gtacatccat acagtgtctg ggtgactgta
     2281 catccataca gcatccacta attttaattc tgctgtaacc cgtacatgcc caaaataggg
     2341 ggcgggttac acagaactgt acatccatac agtgtctggg tgactgtaca tccatacagc
     2401 atccactaaa tataatggag cctgtacatc catacagcat ccactaaaaa aaagaatccc
     2461 agcaccaaaa tattgttttc ttcaccaacc atcagttcat aggtccattc tcttagcgca
     2521 actacagaga acaggggcac aaacaggcaa aaaacgggca caacctcaat ggagtgatgc
     2581 aacctgcctg gagtaaatga tgacacaagg caattgaccc acgcatgtat ctatctcatt
     2641 ttcttacacc ttctattacc ttctgctctc tctgatttgg aaaaagctga aaaaaaaggt
     2701 tgaaaccagt tccctgaaat tattcccctc tgtacatcca tacagtataa atactgtaca
     2761 tccatacagt aattctgtaa atctatttct taaacttctt aaattctact tttatagtta
     2821 gtcttttttt tagttttaaa acaccaagaa cttagtttcg aataaacaca cataaacaaa
     2881 atggattata aagatgacga tgacaaggat tataaagatg acgatgacaa ggattataaa
     2941 gatgacgatg acaagggagg tggtggtggt ggaggcggtc cgacagagaa tcatcgatgg
     3001 gtacccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     3061 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3121 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3181 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3241 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3301 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3361 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3421 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3481 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3541 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3601 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3661 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3721 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3781 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3841 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     3901 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3961 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4021 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4081 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4141 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4201 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4261 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4321 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4381 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4441 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4501 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4561 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4621 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4681 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4741 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4801 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4861 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4921 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4981 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5041 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5101 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5161 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccaccaat acccgactct
     5221 atatgctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     5281 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     5341 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     5401 gactccaacg
//
