LOCUS       sequence-231168-        5267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5267)
  TITLE     Strehler unpublished plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5267)
  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            "Unpublished"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: EGFP-hCaM
FEATURES             Location/Qualifiers
     source          1..5267
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        79..382
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        383..586
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     537..557
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      625..634
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(631..633,634..636,637..1347)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(631..633,634..636,637..1347)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(676..697)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(937..956)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1284..1305
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1363..1389
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1390..1407
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1408..1425
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1483..1932
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPT
                     EAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAA
                     ELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     polyA_signal    2073..2194
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2110..2129)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2164..2183
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2201..2656)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2338..2359)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2550..2569
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2683..2787
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2789..3146
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2789..2980
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2812..2832)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2997..3132
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3181..3975
                     /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             3181..3975
                     /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(3235..3254)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3845..3864
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4163..4182)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4207..4254
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4583..5171
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5072..5091
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag
       61 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      121 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      181 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      241 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      301 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      361 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      421 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      481 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      541 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
      601 tcagatccgc tagcgctacc ggtcgccacc atggtgagca agggcgagga gctgttcacc
      661 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
      721 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
      781 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
      841 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
      901 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
      961 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1021 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1081 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1141 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1201 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     1261 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1321 actctcggca tggacgagct gtacaagtcc ggactcagat cctaccccta tgatgtgcct
     1381 gactatgccc accaccacca ccaccacctg gtgcccaggg gcagcagatc tcgagctcaa
     1441 gcttcgaatt ctgcagtcga cggtacccac aggaaacaga ccatggctga ccagctgact
     1501 gaggagcaga ttgcagagtt caaggaggcc ttctccctct ttgacaagga tggagatggc
     1561 actatcacca ccaaggagtt ggggacagtg atgagatccc tgggacagaa ccccactgaa
     1621 gcagagctgc aggatatgat caatgaggtg gatgcagatg ggaacgggac cattgacttc
     1681 ccggagttcc tgaccatgat ggccagaaag atgaaggaca cagacagtga ggaggagatc
     1741 cgagaggcgt tccgtgtctt tgacaaggat gggaatggct acatcagcgc cgcagagctg
     1801 cgtcacgtaa tgacgaacct gggggagaag ctgaccgatg aggaggtgga tgagatgatc
     1861 agggaggctg acatcgatgg agatggccag gtcaattatg aagagtttgt acagatgatg
     1921 actgcaaagt gaaggccccc cggggatcca ccggatctag ataactgatc ataatcagcc
     1981 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     2041 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     2101 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2161 gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt
     2221 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2281 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2341 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2401 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2461 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     2521 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     2581 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     2641 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2701 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     2761 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     2821 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2881 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     2941 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3001 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3061 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     3121 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     3181 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3241 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3301 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3361 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3421 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3481 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3541 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3601 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3661 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     3721 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3781 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3841 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     3901 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     3961 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4021 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4081 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4141 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     4201 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     4261 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     4321 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     4381 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     4441 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4501 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4561 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4621 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4681 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     4741 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4801 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4861 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4921 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4981 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5041 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5101 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5161 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5221 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataac
//
