LOCUS       sequence-101285-        4889 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 4889)
  TITLE     Directional cloning vector
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4889)
  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            "Unpublished"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-P2A-aGFP
FEATURES             Location/Qualifiers
     source          1..4889
                     /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             736..792
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(937..939,940..1653)
                     /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(937..939,940..1650)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(979..1000)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1240..1259)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1587..1608
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1780..1901
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1817..1836)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1871..1890
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1908..2363)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2045..2066)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2257..2276
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2385..2739
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2385..2576
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2408..2428)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2593..2725
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2655..2674
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2785..3579
                     /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             2785..3579
                     /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(2839..2858)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3449..3468
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3767..3786)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3811..3858
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4187..4775
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4676..4695
                     /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 ccaatgctac taacttcagc ctgctcaagc aggctggaga cgtggaggag
      781 aaccctggac ctgtcgacat taccgattca ctggccgtcg ttctgcaacg ccgcgactgg
      841 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagttgg
      901 cgcaatagcg aagaggcccg caccgaccgc gaattcgtga gcaagggcga ggagctgttc
      961 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1021 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1081 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     1141 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     1201 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1261 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1321 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1381 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     1441 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     1501 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     1561 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1621 atcactctcg gcatggacga gctgtacaag taactcgagt aatctagata actgatcata
     1681 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     1741 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     1801 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     1861 cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt
     1921 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     1981 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     2041 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     2101 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     2161 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     2221 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     2281 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     2341 taatgcgccg ctacagggcg cgtaatattg aaaaaggaag agtcctgagg cggaaagaac
     2401 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     2461 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     2521 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     2581 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     2641 tgactaattt tttttattta tgcagaggcc gaggccgcgg cctctgagct attccagaag
     2701 tagtgaggag gcttttttgg aggcctaggc ttttgcaaat tcaaatattt atccgctcat
     2761 aagacaatcc aagaaggagc caccatgatt gaacaagatg gattgcacgc aggttctccg
     2821 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     2881 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     2941 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg
     3001 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3061 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     3121 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     3181 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     3241 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     3301 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     3361 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     3421 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     3481 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     3541 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     3601 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     3661 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     3721 gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg
     3781 agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg
     3841 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata
     3901 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc
     3961 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca
     4021 tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4081 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4141 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4201 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4261 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4321 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4381 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4441 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4501 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4561 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4621 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4681 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4741 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4801 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4861 ctgtggataa ccgtattacc gccatgcat
//
