LOCUS       sequence-7029-ex        5195 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 5195)
  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 5195)
  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: V954 - pDNR MCS SA no SA CmR - Donor
FEATURES             Location/Qualifiers
     source          1..5195
                     /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    206..227
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(242..247,248..265,266..272)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    280..296
                     /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     285..307
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     304..320
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     304..320
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             316..489
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     complement(336..352)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(345..367)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      complement(546..640)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(661..1320)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1235..1254
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     protein_bind    complement(1349..1382)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1397..1842
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(1843..1929,1930..3264)
                     /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        3332..3436
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3437..3505,3506..4297)
                     /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(3655..3674)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4468..5056
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4957..4976
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5173..5189
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ataacttcgt atagcataca ttatacgaag ttatggcgcg agggtttcct tgacaatatc
       61 atacttatcc tgtccctttt ttttccccag gggcgcgccg gcgcccaata cgcaaaccgc
      121 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
      181 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
      241 ctttacactt tatgcttccg gctcgtatgt tgtgtgaaat tgtgagcgga taacaatttc
      301 acacaggaaa cagctatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt
      361 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
      421 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
      481 aatggcgaat ggcgttaatt aaccaggtaa gttcctagga gatcctggtc atgactagtg
      541 cttggattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa caaatccaga
      601 tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct cgatatcaaa
      661 ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac
      721 atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc
      781 gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc
      841 cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt
      901 ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga
      961 atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt
     1021 ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc
     1081 accgtctttc attgccatac gaaattccgg atgagcattc atcaggcggg caagaatgtg
     1141 aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat
     1201 atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg
     1261 ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat
     1321 tttggcttcc ttggctcctg aaagatccat aacttcgtat agcatacatt atacgaagtt
     1381 atgcggccgc gacgtccaca tatacctgcc gttcactatt atttagtgaa atgagatatt
     1441 atgatatttt ctgaattgtg attaaaaagg caactttatg cccatgcaac agaaactata
     1501 aaaaatacag agaatgaaaa gaaacagata gattttttag ttctttaggc ccgtagtctg
     1561 caaatccttt tatgattttc tatcaaacaa aagaggaaaa tagaccagtt gcaatccaaa
     1621 cgagagtcta atagaatgag gtcgaaaagt aaatcgcgcg ggtttgttac tgataaagca
     1681 ggcaagacct aaaatgtgta aagggcaaag tgtatacttt ggcgtcaccc cttacatatt
     1741 ttaggtcttt ttttattgtg cgtaactaac ttgccatctt caaacaggag ggctggaaga
     1801 agcagaccgc taacacagta cataaaaaag gagacatgaa cgatgaacat caaaaagttt
     1861 gcaaaacaag caacagtatt aacctttact accgcactgc tggcaggagg cgcaactcaa
     1921 gcgtttgcga aagaaacgaa ccaaaagcca tataaggaaa catacggcat ttcccatatt
     1981 acacgccatg atatgctgca aatccctgaa cagcaaaaaa atgaaaaata tcaagttcct
     2041 gagttcgatt cgtccacaat taaaaatatc tcttctgcaa aaggcctgga cgtttgggac
     2101 agctggccat tacaaaacgc tgacggcact gtcgcaaact atcacggcta ccacatcgtc
     2161 tttgcattag ccggagatcc taaaaatgcg gatgacacat cgatttacat gttctatcaa
     2221 aaagtcggcg aaacttctat tgacagctgg aaaaacgctg gccgcgtctt taaagacagc
     2281 gacaaattcg atgcaaatga ttctatccta aaagaccaaa cacaagaatg gtcaggttca
     2341 gccacattta catctgacgg aaaaatccgt ttattctaca ctgatttctc cggtaaacat
     2401 tacggcaaac aaacactgac aactgcacaa gttaacgtat cagcatcaga cagctctttg
     2461 aacatcaacg gtgtagagga ttataaatca atctttgacg gtgacggaaa aacgtatcaa
     2521 aatgtacagc agttcatcga tgaaggcaac tacagctcag gcgacaacca tacgctgaga
     2581 gatcctcact acgtagaaga taaaggccac aaatacttag tatttgaagc aaacactgga
     2641 actgaagatg gctaccaagg cgaagaatct ttatttaaca aagcatacta tggcaaaagc
     2701 acatcattct tccgtcaaga aagtcaaaaa cttctgcaaa gcgataaaaa acgcacggct
     2761 gagttagcaa acggcgctct cggtatgatt gagctaaacg atgattacac actgaaaaaa
     2821 gtgatgaaac cgctgattgc atctaacaca gtaacagatg aaattgaacg cgcgaacgtc
     2881 tttaaaatga acggcaaatg gtacctgttc actgactccc gcggatcaaa aatgacgatt
     2941 gacggcatta cgtctaacga tatttacatg cttggttatg tttctaattc tttaactggc
     3001 ccatacaagc cgctgaacaa aactggcctt gtgttaaaaa tggatcttga tcctaacgat
     3061 gtaaccttta cttactcaca cttcgctgta cctcaagcga aaggaaacaa tgtcgtgatt
     3121 acaagctata tgacaaacag aggattctac gcagacaaac aatcaacgtt tgcgcctagc
     3181 ttcctgctga acatcaaagg caagaaaaca tctgttgtca aagacagcat ccttgaacaa
     3241 ggacaattaa cagttaacaa ataaaaacgc aaaagaaaat gccgatatcc tattggcatt
     3301 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3361 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3421 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3481 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3541 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3601 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     3661 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     3721 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     3781 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     3841 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     3901 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     3961 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     4021 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     4081 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     4141 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     4201 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     4261 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     4321 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4381 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4441 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4501 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4561 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4621 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4681 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4741 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4801 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4861 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4921 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4981 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5041 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5101 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5161 cgccttacgc gtgtaaaacg acggccagta gatct
//
