LOCUS       sequence-174745-        5478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET LIC cloning vector for yORF-mClover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5478)
  TITLE     C-terminal fluorescent fusion vectors for E. coli expression
REFERENCE   2  (bases 1 to 5478)
  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"
COMMENT     Sequence Label: pET mClover LIC cloning vector (u-mClover)
FEATURES             Location/Qualifiers
     source          1..5478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1499..1518
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1602..1621
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1602..1620
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1709..1731
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(1772..1774,1775..2485)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1814..1835)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2422..2443
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2609..2627)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2623..2670
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3036..3041,3042..3058,3059..3064))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3091..3109)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3177..3281
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3282..3350,3351..4142)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3500..3519)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4313..4901
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4802..4821
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5055..5072
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5087..5226
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5152..5171)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5312..5334
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(5328..5478,1..41))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
       61 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
      121 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
      181 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
      241 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
      301 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
      361 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
      421 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
      481 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc
      541 gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca
      601 ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg
      661 aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg
      721 cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc
      781 tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg
      841 taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca
      901 gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga
      961 aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca
     1021 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     1081 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     1141 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     1201 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     1261 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgacgctct cccttatgcg
     1321 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     1381 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     1441 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     1501 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     1561 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
     1621 gagaccacaa cggtttccct ctagtgccgg ctccggagag ctctttaatt aagcggccgc
     1681 cctgcaggac tcgagttcta gaaataattt tgtttaactt taagaaggag atatagatat
     1741 cctctcatcc tccaacggca gcagcggatc cgtgagcaag ggcgaggagc tgttcaccgg
     1801 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtccg
     1861 cggcgagggc gagggcgatg ccaccaacgg caagctgacc ctgaagttca tctgcaccac
     1921 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcgtggcctg
     1981 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2041 aggctacgtc caggagcgca ccatctcttt caaggacgac ggtacctaca agacccgcgc
     2101 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2161 caaggaggac ggcaacatcc tggggcacaa gctggagtac aacttcaaca gccacaacgt
     2221 ctatatcacg gccgacaagc agaagaacgg catcaaggct aacttcaaga tccgccacaa
     2281 cgttgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     2341 cggccccgtg ctgctgcccg acaaccacta cctgagccat cagtccgccc tgagcaaaga
     2401 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggattac
     2461 acatggcatg gacgagctgt acaagtagta accaactcca taaggatccg cgatcgcggc
     2521 gcgccacctg gtggccggcc ggtaccacgc gtgcgcgctg atccggctgc taacaaagcc
     2581 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     2641 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggacatcca
     2701 caggacgggt gtggtcgcca tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag
     2761 caggactggg cggcggccaa agcggtcgga cagtgctccg agaacgggtg cgcatagaaa
     2821 ttgcatcaac gcatatagcg ctagcagcac gccatagtga ctggcgatgc tgtcggaatg
     2881 gacgacatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc
     2941 cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg
     3001 actgcgttag caatttaact gtgataaact accgcattaa agcttatcga tgataagctg
     3061 tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
     3121 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     3181 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3241 aaccctgata aatgcttcaa taacattgaa aaaggaagag tatgagtatt caacatttcc
     3301 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     3361 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     3421 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     3481 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     3541 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     3601 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     3661 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     3721 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     3781 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     3841 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     3901 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     3961 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     4021 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     4081 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     4141 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     4201 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     4261 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4321 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4381 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4441 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4501 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4561 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4621 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4681 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4741 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4801 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4861 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4921 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4981 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     5041 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     5101 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     5161 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     5221 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5281 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5341 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     5401 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     5461 aatgtctggc ttctgata
//
