LOCUS       pKG2436_pGEEC532        4852 bp    DNA     circular SYN 09-OCT-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4852)
  AUTHORS   ISU Biology IT
  TITLE     Direct Submission
  JOURNAL   Exported May 7, 2025 from SnapGene 8.0.3
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..42
                     /label=oMJ001
     primer_bind     1..42
                     /label=oMJ005
     primer_bind     3..42
                     /label=oMJ009
     primer_bind     3..42
                     /label=oMJ013
     primer_bind     9..39
                     /label=oMJ020
     primer_bind     complement(21..46)
                     /label=oSEQ033 Pentr_rev
     misc_feature    23..46
                     /label=pENTR2B.Seq.REV
     primer_bind     complement(35..56)
                     /label=oTA069 mIR-backbone_junction_rev
     primer_bind     complement(35..56)
                     /label=oSEQ044 pENTR_rev_seq
     primer_bind     complement(108..128)
                     /label=oSEQ005 pENTR_rev
                     /note="25nm	STD"
     CDS             122..931
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(147..168)
                     /label=oSEQ023 attl2_rev
     primer_bind     complement(155..178)
                     /label=Kan_rev_SEQ
     primer_bind     complement(545..574)
                     /label=oTA099 GG_entry_upstream_fwd
     primer_bind     545..574
                     /label=oTA102 GG_entry_downstream_rev
     misc_feature    1005..1624
                     /label=pBR322_Origin
     rep_origin      1021..1609
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1416..1431
                     /label=oTA073 Xho_addition_rev
     primer_bind     complement(1486..1501)
                     /label=oTA072 Xho_addition_for
     primer_bind     1510..1529
                     /label=oSEQ067 pBR322-ori_fwd
     primer_bind     1761..1774
                     /label=oSEQ011 pEntr_for
                     /note="25nm	STD"
     terminator      1774..1860
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1923..1942
                     /label=oSEQ011 pEntr_for
                     /note="25nm	STD"
     terminator      1952..1979
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     1981..2000
                     /label=pENTR_fwd_seq
     primer_bind     1981..2000
                     /label=oSEQ043 pENTR_fwd_seq
     primer_bind     complement(2002..2046)
                     /label=oMJ002
     primer_bind     complement(2002..2042)
                     /label=oMJ010
     primer_bind     complement(2002..2038)
                     /label=oMJ006
     primer_bind     complement(2002..2038)
                     /label=oMJ014
     primer_bind     complement(2010..2038)
                     /label=oMJ019
     promoter        2047..3228
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     primer_bind     complement(2107..2124)
                     /label=oSEQ045 phage-dest-rev-2
     intron          2277..3219
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3015..3036)
                     /label=oSEQ046 phage-dest-upstream-seq
     primer_bind     3079..3098
                     /label=oSEQ064 EF1a_fwd
     primer_bind     complement(3210..3245)
                     /label=oEP002
     primer_bind     complement(3210..3233)
                     /label=oEP142
     primer_bind     complement(3210..3229)
                     /label=oEP008
     primer_bind     3234..3248
                     /label=oKL260
     regulatory      3234..3243
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3240..3950
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     primer_bind     3240..3257
                     /label=oCO03 mRuby2_fwd
     primer_bind     3240..3257
                     /label=oKL266
     primer_bind     complement(3266..3287)
                     /label=oEP076
     misc_feature    3286..3289
                     /label=splice site 1
     primer_bind     3288..3307
                     /label=oEP073
     primer_bind     3310..3329
                     /label=oSEQ062 mRuby2_fwd
     primer_bind     complement(3310..3329)
                     /label=oSEQ093 mRuby2_rev
     misc_feature    3508..3511
                     /label=splice site 2
     primer_bind     complement(3728..3747)
                     /label=oCO04 mRuby2_rev
     misc_feature    3786..3789
                     /label=splice site 3
     primer_bind     complement(3842..3861)
                     /label=oSEQ061 mRuby2_rev
     primer_bind     3842..3859
                     /label=oKL117
     primer_bind     3876..3895
                     /label=OSEQ091 mRuby2_fwd_SEQ
     primer_bind     complement(3930..3971)
                     /label=oKL303
                     /note="rev for PCR192"
     primer_bind     3951..3973
                     /label=oKL166
     primer_bind     3951..3972
                     /label=oKL141
     primer_bind     3951..3971
                     /label=oKL183
     primer_bind     complement(3951..3970)
                     /label=oKL182
     CDS             3960..4016
                     /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"
     primer_bind     3997..4036
                     /label=oKL250
     primer_bind     3997..4019
                     /label=oKL266
     primer_bind     complement(3999..4016)
                     /label=oKL202
     CDS             4017..4616
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     4017..4038
                     /label=pPV2-puro_fwd
     primer_bind     complement(4017..4036)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4017..4036
                     /label=oSEQ047 phage-dest-downtream-seq
     primer_bind     complement(4482..4509)
                     /label=puro1_rev
     primer_bind     4490..4517
                     /label=puro2_fwd
     primer_bind     4513..4533
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4582..4597
                     /label=OSEQ086 Puro_rev_SEQ
     primer_bind     complement(4596..4614)
                     /label=oKL300
                     /note="for PCR188 P2A-PuroR"
     primer_bind     complement(4596..4613)
                     /label=oKL066
     primer_bind     complement(4596..4613)
                     /label=oKL251
     primer_bind     4599..4640
                     /label=gebGH_fwd
     primer_bind     complement(4599..4620)
                     /label=pPV2-puro_rev
     primer_bind     4622..4643
                     /label=GG_bGH_fwd
     primer_bind     4626..4643
                     /label=GG_WPRE-bGH_fwd
     polyA_signal    4628..4852
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(join(4834..4852,1..4))
                     /label=gebGH_rev
     primer_bind     complement(4838..4852)
                     /label=GG_bGH_rev
ORIGIN
        1 cgctcgctga ctgcaggtgc ccgtgtctca aaatctctga tgttacattg cacaagataa
       61 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
      121 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
      181 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
      241 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
      301 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
      361 gaccatcaag cattttatcc gtactcctgg tgatgcatgg ttactcacca ctgcgatccc
      421 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
      481 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
      541 cagcgatcgc gtatttcgtc tagctcaggc gcaatcacga atgaataacg gtttggttga
      601 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
      661 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
      721 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
      781 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
      841 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
      901 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacatta
      961 ttcagattgg gccccgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     1021 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     1081 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     1141 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     1201 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1261 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1321 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     1381 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     1441 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     1501 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     1561 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     1621 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     1681 gttatcccct gattctgtgg ataaccgtat taccgctagc atggatctcg gggacgtcta
     1741 actactaagc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcggaa
     1801 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     1861 ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg ggcaggacgc
     1921 ccgccataaa ctgccaggca tcaaactaag cagaaggcca tcctgacgga tggccttttt
     1981 gcgtttctac aaactcttcc tgttagttag ttacttaagc tcgggcccac ctgcagtcta
     2041 cttactgctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
     2101 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg
     2161 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     2221 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa
     2281 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt
     2341 gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     2401 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     2461 gaggcctggc ttgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     2521 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     2581 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
     2641 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
     2701 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     2761 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     2821 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     2881 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     2941 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     3001 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt
     3061 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     3121 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     3181 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaga atggccacca
     3241 tggtgtctaa gggcgaagag ctgatcaagg aaaatatgcg tatgaaggtg gtcatggaag
     3301 gttcggtcaa cggccaccaa ttcaaatgca caggtgaagg agaaggcaat ccgtacatgg
     3361 gaactcaaac catgaggatc aaagtcatcg agggaggacc cctgccattt gcctttgaca
     3421 ttcttgccac gtcgttcatg tatggcagcc gtacttttat caagtacccg aaaggcattc
     3481 ctgatttctt taaacagtcc tttcctgagg gttttacttg ggaaagagtt acgagatacg
     3541 aagatggtgg agtcgtcacc gtcatgcagg acaccagcct tgaggatggc tgtctcgttt
     3601 accacgtcca agtcagaggg gtaaactttc cctccaatgg tcccgtgatg cagaagaaga
     3661 ccaagggttg ggagcctaat acagagatga tgtatccagc agatggtggt ctgaggggat
     3721 acactcatat ggcactgaaa gttgatggtg gtggccatct gtcttgctct ttcgtaacaa
     3781 cttacaggtc aaaaaagacc gtcgggaaca tcaagatgcc cggtatccat gccgttgatc
     3841 accgcctgga aaggttagag gaaagtgaca atgaaatgtt cgtagtacaa cgcgaacacg
     3901 cagttgccaa gttcgccggg cttggtggtg ggatggacga gctgtacaag ggaagcggag
     3961 ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctatga
     4021 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     4081 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     4141 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     4201 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     4261 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     4321 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     4381 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     4441 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     4501 catccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     4561 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacaac
     4621 gttgcacctg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     4681 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     4741 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     4801 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat gg
//
