LOCUS       sequence-263326-        5256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5256)
  AUTHORS   Wassmann K, Liberal V, Benezra R
  TITLE     Mad2 phosphorylation regulates its association with Mad1 and the 
            APC/C.
  JOURNAL   EMBO J. 2003 Feb 17. 22(4):797-806.
  PUBMED    12574116
REFERENCE   2  (bases 1 to 5256)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "EMBO J. 
            2003 Feb 17. 22(4):797-806."
COMMENT     Sequence Label: VL3 (Flag-Mad2)
FEATURES             Location/Qualifiers
     source          1..5256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     126..148
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     140..157
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     852..872
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             935..958
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    1597..2219
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1796..1815)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2243..2263)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2248..2577
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2428..2563
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2615..2634)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2616..2634)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2648..2664)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2648..2664)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2661..2683)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2672..2688
                     /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(join(2696..2702,2703..2720,2721..2726))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2741..2762
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2879..2896)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3050..3638)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3130..3149)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3809..4600,4601..4669))
                     /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"
     primer_bind     4432..4451
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4670..4774)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4801..5229
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4888..4907)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5098..5119
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
       61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa
      181 tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta
      241 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      301 aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
      361 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      421 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      481 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      541 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      601 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      661 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      721 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      781 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      841 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      901 tcgtttagtg aaccgtcaga attgatctac catggactac aaagacgatg acgacaagct
      961 taccatggcg ctgcagctct cccgggagca gggaatcacc ctgcgcggga gcgccgaaat
     1021 cgtggccgag ttcttctcat tcggcatcaa cagcatttta tatcagcgtg gcatatatcc
     1081 atctgaaacc tttactcgag tgcagaaata cggactcacc ttgcttgtaa ctactgatct
     1141 tgagctcata aaatacctaa ataatgtggt ggaacaactg aaagattggt tatacaagtg
     1201 ttcagttcag aaactggttg tagttatctc aaatattgaa agtggtgagg tcctggaaag
     1261 atggcagttt gatattgagt gtgacaagac tgcaaaagat gacagtgcac ccagagaaaa
     1321 gtctcagaaa gctatccagg atgaaatccg ttcagtgatc agacagatca cagctacggt
     1381 gacatttctg ccactgttgg aagtttcttg ttcatttgat ctgctgattt atacagacaa
     1441 agatttggtt gtacctgaaa aatgggaaga gtcgggacca cagtttatta ccaattctga
     1501 ggaagtccgc cttcgttcat ttactactac aatccacaaa gtaaatagca tggtggccta
     1561 caaaattcct gtcaatgact gaggtaccag gatcccgggt ggcatccctg tgacccctcc
     1621 ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata
     1681 aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg
     1741 ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat
     1801 tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg
     1861 gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc
     1921 aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg
     1981 gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta
     2041 caggcgtgaa ccactgctcc cttccctgtc cttctgattt taaaataact ataccagcag
     2101 gaggacgtcc agacacagca taggctacct ggccatgccc aaccggtggg acatttgagt
     2161 tgcttgcttg gcactgtcct ctcatgcgtt gggtccactc agtagatgcc tgttgaattg
     2221 ggtacgcggc cagcttggct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2281 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2341 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2401 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2461 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     2521 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     2581 ctcctcgagg aactgaaaaa ccagaaagtt aattccctat agtgagtcgt attaaattcg
     2641 taatcatgtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2701 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2761 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2821 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2881 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2941 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3001 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3061 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3121 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3181 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3241 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3301 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3361 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3421 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3481 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3541 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3601 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3661 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3721 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3781 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3841 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3901 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3961 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4021 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4081 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4141 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4201 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4261 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4321 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4381 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4441 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4501 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4561 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4621 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4681 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4741 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4801 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4861 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4921 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4981 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     5041 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5101 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     5161 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5221 acgcgaattt taacaaaata ttaacgctta caattt
//
