LOCUS       sequence-482022-        3518 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3518)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3518)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: SA-EGFP-pA
FEATURES             Location/Qualifiers
     source          1..3518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(1..87)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     136..158
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     150..167
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     151..167
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      272..281
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(278..280,281..283,284..997)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSAQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(323..344)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(584..603)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     931..952
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1027..1161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1051..1070)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1105..1124
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1598..2389
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      2482..3070
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2971..2990
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3224..3241
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3400..3427)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
       61 ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcgttaacgc
      121 tagcatggat gttttcccag tcacgacgtt gtaaaacgac ggccagtctt aagctcgggc
      181 ccggtctctt cgccactaat ccggagtgtg tgtgtgtttc tgacgtgtct ctctcttccc
      241 tttttttttt cagacggctg atttttctaa cgccaccatg gtgagcaagg gcgaggagct
      301 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
      361 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
      421 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
      481 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
      541 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
      601 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
      661 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
      721 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
      781 ccgccacaac atcgaggacg gcagcgcgca gctcgccgac cactaccagc agaacacccc
      841 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
      901 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
      961 cgggatcact ctcggcatgg acgagctgta caagtaagct agaactatag tgagtcgtat
     1021 tacgtagatc cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1081 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1141 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     1201 ggggaggtgt gggaggtttt ttaattcctc aagagacctc tagaccagcc aggacagaaa
     1261 tgcctcgact tcgctgctac ccaaggttgc cgggtgacgc acaccgtgga aacggatgaa
     1321 ggcacgaacc cagtggacat aagcctgttc ggttcgtaag ctgtaatgca agtagcgtat
     1381 gcgctcacgc aactggtcca gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc
     1441 ggttttcatg gcttgttatg actgtttttt tggggtacag tctatgcctc gggcatccaa
     1501 gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta tggagcagca acgatgttac
     1561 gcagcagggc agtcgcccta aaacaaagtt aaacattatg agggaagcgg tgatcgccga
     1621 agtatcgact caactatcag aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt
     1681 gctggccgta catttgtacg gctccgcagt ggatggcggc ctgaagccac acagtgatat
     1741 tgatttgctg gttacggtga ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa
     1801 cgaccttttg gaaacttcgg cttcccctgg agagagcgag attctccgcg ctgtagaagt
     1861 caccattgtt gtgcacgacg acatcattcc gtggcgttat ccagctaagc gcgaactgca
     1921 atttggagaa tggcagcgca atgacattct tgcaggtatc ttcgagccag ccacgatcga
     1981 cattgatctg gctatcttgc tgacaaaagc aagagaacat agcgttgcct tggtaggtcc
     2041 agcggcggag gaactctttg atccggttcc tgaacaggat ctatttgagg cgctaaatga
     2101 aaccttaacg ctatggaact cgccgcccga ctgggctggc gatgagcgaa atgtagtgct
     2161 tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc
     2221 tgccgactgg gcaatggagc gcctgccggc ccagtatcag cccgtcatac ttgaagctag
     2281 acaggcttat cttggacaag aagaagatcg cttggcctcg cgcgcagatc agttggaaga
     2341 atttgtccac tacgtgaaag gcgagatcac caaggtagtc ggcaaataac cctcgagcca
     2401 cccatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt
     2461 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2521 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2581 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     2641 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2701 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2761 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2821 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2881 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2941 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3001 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3061 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3121 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3181 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3241 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     3301 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     3361 tacgcgtacc gctagccagg aagagtttgt agaaacgcaa aaaggccatc cgtcaggatg
     3421 gccttctgct tagtttgatg cctggcagtt tatggcgggc gtcctgcccg ccaccctccg
     3481 ggccgttgct tcacaacgtt caaatccgct cccggcgg
//
