LOCUS       sequence-101282-        3789 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 3789)
  TITLE     Directional cloning vector
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3789)
  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            "Unpublished"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: pUC-dirTopo-P2A-aGFP
FEATURES             Location/Qualifiers
     source          1..3789
                     /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"
     promoter        1951..2055
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2056..2124,2125..2916)
                     /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(2274..2293)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3087..3675
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3576..3595
                     /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 taatcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1981 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2041 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2101 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2161 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2221 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2281 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     2341 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2401 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2461 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2521 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2581 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2641 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2701 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2761 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2821 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2881 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     2941 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3001 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3061 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3121 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3181 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     3241 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3301 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3361 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3421 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3481 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3541 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3601 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3661 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3721 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3781 gccatgcat
//
