LOCUS       sequence-458091-        5241 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG promoter-driven expression vector suitable for ligation of 
            multiple inserts by Golden Gate Cloning using Esp3I.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5241)
  AUTHORS   Hermann M, Stillhard P, Wildner H, Seruggia D, Kapp V, 
            Sanchez-Iranzo H, Mercader N, Montoliu L, Zeilhofer HU, Pelczar P
  TITLE     Binary recombinase systems for high-resolution conditional 
            mutagenesis.
  JOURNAL   Nucleic Acids Res. 2014 Jan 9.
  PUBMED    24413561
REFERENCE   2  (bases 1 to 5241)
  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: "Nucleic 
            Acids Res. 2014 Jan 9."
COMMENT     Sequence Label: pCAG-Golden-Gate-Esp3I-Destination
FEATURES             Location/Qualifiers
     source          1..5241
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        9..388
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..667
                     /label=chicken beta-actin promoter
     intron          668..1684
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1608..1630
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        1697..1715
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(1709..2014)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLRWRVDPL"
     primer_bind     1879..1895
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1905..1923
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1971..1991)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1971..1989)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2010..2026)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2034..2050
                     /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(2058..2064,2065..2082,2083..2088))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2103..2124
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2163..2180)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2169..2393
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2465..2481)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(2622..3077)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2759..2780)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2971..2990
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3104..3208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3209..3277,3278..4069)
                     /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(3427..3446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4240..4828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4729..4748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4982..4999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5116..5137
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5152..5157,5158..5175,5176..5182)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5190..5206
                     /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     5214..5230
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ccggattcga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
       61 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      121 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      181 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      241 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      301 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      361 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
      421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
      481 tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga
      541 ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg
      601 aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg
      661 gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg
      721 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc
      781 ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct
      841 gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg
      901 tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg
      961 tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc
     1021 ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg
     1081 gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc
     1141 cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac
     1201 ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg
     1261 cggggcgggg ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag
     1321 cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga
     1381 gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg
     1441 ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg
     1501 cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg
     1561 ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg
     1621 gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct
     1681 acagatcctt aattaataat acgactcact atagggggtc gacccgccac cggagacgcc
     1741 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     1801 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     1861 tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat
     1921 agggcgaatt gggtaccggg ccccccctcg aggtcctcca gcttttgttc cctttagtga
     1981 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     2041 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     2101 taatgagtga gctaactcac attaattgcg ttgcgccgtc tccgtcagag ctcgctgatc
     2161 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2221 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2281 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2341 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2401 ggcggaaaga accagctggg gctcgagatc cactagttct agcctcgagg ctagagcggc
     2461 cgccactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     2521 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     2581 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gaaattgtaa gcgttaatat
     2641 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     2701 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     2761 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     2821 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     2881 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     2941 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     3001 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     3061 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3181 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     3241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3421 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     3481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     3541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     3601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3721 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     3781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     3901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     3961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     4021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     4081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4381 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4981 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     5041 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     5101 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     5161 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     5221 cagctatgac catgaggcgc g
//
