LOCUS       sequence-101286-        4850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A directional cloning vector of PCR-amplified cDNAs for mammalian 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4850)
  TITLE     Directional cloning vector
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4850)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCMVlac-dirTopo-AU1-aGFP
FEATURES             Location/Qualifiers
     source          1..4850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        367..570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     521..541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    637..653
                     /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)."
     CDS             join(898..900,901..1614)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(898..900,901..1611)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(940..961)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1201..1220)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1548..1569
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1741..1862
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1778..1797)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1832..1851
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1869..2324)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2006..2027)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2218..2237
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2346..2700
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2346..2537
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2369..2389)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2554..2686
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2616..2635
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2746..3540
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2746..3540
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2800..2819)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3410..3429
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3728..3747)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3772..3819
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4148..4736
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4637..4656
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      421 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      481 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      541 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagctt
      601 tacactttat tcttccggct cgtattttgt gtggaattgt gagcggataa caatttccgg
      661 aggaggccct tcacctgaag agcggacgga cgagcggata tcgccgccta accccgctct
      721 tcaaagggat ccaatgacac ctacaggtac atcgtcgaca ttaccgattc actggccgtc
      781 gttctgcaac gccgcgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
      841 catccccctt tcgccagttg gcgcaatagc gaagaggccc gcaccgaccg cgaattcgtg
      901 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      961 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1021 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1081 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     1141 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1201 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1261 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1321 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     1381 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1441 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1501 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1561 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaactcgag
     1621 taatctagat aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta
     1681 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     1741 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     1801 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     1861 taacgcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     1921 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     1981 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     2041 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     2101 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     2161 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     2221 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     2281 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaatatt gaaaaaggaa
     2341 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     2401 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     2461 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     2521 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     2581 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcg
     2641 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     2701 ttcaaatatt tatccgctca taagacaatc caagaaggag ccaccatgat tgaacaagat
     2761 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     2821 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     2881 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     2941 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3001 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3061 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3121 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3181 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     3241 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     3301 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     3361 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     3421 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     3481 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     3541 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     3601 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     3661 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     3721 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     3781 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     3841 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     3901 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     3961 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
     4021 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     4081 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4141 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4201 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4261 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     4321 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4381 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4441 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4501 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4561 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4621 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4681 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4741 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4801 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
