LOCUS       sequence-7026-ex        5177 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 5177)
  AUTHORS   Colwill K, Wells CD, Elder K, Goudreault M, Hersi K, Kulkarni S, 
            Hardy WR, Pawson T, Morin GB
  TITLE     Modification of the Creator recombination system for proteomics 
            applications--improved expression by addition of splice sites.
  JOURNAL   BMC Biotechnol. 2006 Mar 6;6:13. doi: 10.1186/1472-6750-6-13.
  PUBMED    16519801
REFERENCE   2  (bases 1 to 5177)
  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: "BMC 
            Biotechnol."; date: "2006-03-6"; volume: "6:13. doi"; pages: " 
            10.1186/1472-6750-6-13"
COMMENT     Sequence Label: V677 pDNR MCS BE lacZ alpha - Donor
FEATURES             Location/Qualifiers
     source          1..5177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(1..34)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    155..176
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(191..196,197..214,215..221)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    229..245
                     /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     234..256
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     253..269
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     253..269
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             265..438
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     complement(285..301)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(294..316)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      complement(500..594)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(615..1274)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1189..1208
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     protein_bind    complement(1303..1336)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1351..1796
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(1797..1883,1884..3218)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        3286..3390
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3391..3459,3460..4251)
                     /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     complement(3609..3628)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4422..5010
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4911..4930
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5127..5143
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5151..5170
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5151..5169
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ataacttcgt atagcataca ttatacgaag ttatggggat ccgtcgacgg cgcccaatac
       61 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
      121 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
      181 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtgaaatt gtgagcggat
      241 aacaatttca cacaggaaac agctatgacc atgattacgg attcactggc cgtcgtttta
      301 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
      361 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
      421 cgcagcctga atggcgaatg gcgaagcttg aattccaggt aagttagcct aggagatcct
      481 ggtcatgact agtgcttgga ttctcaccaa taaaaaacgc ccggcggcaa ccgagcgttc
      541 tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg agtccaagcg
      601 agctcgatat caaattacgc cccgccctgc cactcatcgc agtactgttg taattcatta
      661 agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat cgccagcggc
      721 atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag
      781 ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag
      841 acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac
      901 gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag
      961 agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc
     1021 catatcacca gctcaccgtc tttcattgcc atacgaaatt ccggatgagc attcatcagg
     1081 cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt
     1141 aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga
     1201 aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg
     1261 atttttttct ccattttagc ttccttagct cctgaaagat ccataacttc gtatagcata
     1321 cattatacga agttatgcgg ccgcgacgtc cacatatacc tgccgttcac tattatttag
     1381 tgaaatgaga tattatgata ttttctgaat tgtgattaaa aaggcaactt tatgcccatg
     1441 caacagaaac tataaaaaat acagagaatg aaaagaaaca gatagatttt ttagttcttt
     1501 aggcccgtag tctgcaaatc cttttatgat tttctatcaa acaaaagagg aaaatagacc
     1561 agttgcaatc caaacgagag tctaatagaa tgaggtcgaa aagtaaatcg cgcgggtttg
     1621 ttactgataa agcaggcaag acctaaaatg tgtaaagggc aaagtgtata ctttggcgtc
     1681 accccttaca tattttaggt ctttttttat tgtgcgtaac taacttgcca tcttcaaaca
     1741 ggagggctgg aagaagcaga ccgctaacac agtacataaa aaaggagaca tgaacgatga
     1801 acatcaaaaa gtttgcaaaa caagcaacag tattaacctt tactaccgca ctgctggcag
     1861 gaggcgcaac tcaagcgttt gcgaaagaaa cgaaccaaaa gccatataag gaaacatacg
     1921 gcatttccca tattacacgc catgatatgc tgcaaatccc tgaacagcaa aaaaatgaaa
     1981 aatatcaagt tcctgagttc gattcgtcca caattaaaaa tatctcttct gcaaaaggcc
     2041 tggacgtttg ggacagctgg ccattacaaa acgctgacgg cactgtcgca aactatcacg
     2101 gctaccacat cgtctttgca ttagccggag atcctaaaaa tgcggatgac acatcgattt
     2161 acatgttcta tcaaaaagtc ggcgaaactt ctattgacag ctggaaaaac gctggccgcg
     2221 tctttaaaga cagcgacaaa ttcgatgcaa atgattctat cctaaaagac caaacacaag
     2281 aatggtcagg ttcagccaca tttacatctg acggaaaaat ccgtttattc tacactgatt
     2341 tctccggtaa acattacggc aaacaaacac tgacaactgc acaagttaac gtatcagcat
     2401 cagacagctc tttgaacatc aacggtgtag aggattataa atcaatcttt gacggtgacg
     2461 gaaaaacgta tcaaaatgta cagcagttca tcgatgaagg caactacagc tcaggcgaca
     2521 accatacgct gagagatcct cactacgtag aagataaagg ccacaaatac ttagtatttg
     2581 aagcaaacac tggaactgaa gatggctacc aaggcgaaga atctttattt aacaaagcat
     2641 actatggcaa aagcacatca ttcttccgtc aagaaagtca aaaacttctg caaagcgata
     2701 aaaaacgcac ggctgagtta gcaaacggcg ctctcggtat gattgagcta aacgatgatt
     2761 acacactgaa aaaagtgatg aaaccgctga ttgcatctaa cacagtaaca gatgaaattg
     2821 aacgcgcgaa cgtctttaaa atgaacggca aatggtacct gttcactgac tcccgcggat
     2881 caaaaatgac gattgacggc attacgtcta acgatattta catgcttggt tatgtttcta
     2941 attctttaac tggcccatac aagccgctga acaaaactgg ccttgtgtta aaaatggatc
     3001 ttgatcctaa cgatgtaacc tttacttact cacacttcgc tgtacctcaa gcgaaaggaa
     3061 acaatgtcgt gattacaagc tatatgacaa acagaggatt ctacgcagac aaacaatcaa
     3121 cgtttgcgcc tagcttcctg ctgaacatca aaggcaagaa aacatctgtt gtcaaagaca
     3181 gcatccttga acaaggacaa ttaacagtta acaaataaaa acgcaaaaga aaatgccgat
     3241 atcctattgg cattgacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3301 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3361 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3421 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3481 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3541 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3601 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3661 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3721 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     3781 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3841 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3901 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3961 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4021 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4081 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4141 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4201 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4261 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4321 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4381 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4441 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4501 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4561 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4621 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4681 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4741 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4801 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4861 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4921 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4981 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5041 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5101 gataaccgta ttaccgcctt acgcgtgtaa aacgacggcc agtagatctg taatacgact
     5161 cactataggg cgctagc
//
