LOCUS       pKG2441_pGEEC537        5379 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 5379)
  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..5379
                     /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        2048..2049
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     enhancer        2052..2412
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(2280..2299)
                     /label=oSEQ065 CMV_rev
     promoter        2414..2691
                     /label=chicken beta-actin promoter
     intron          2692..3708
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3723..3742
                     /label=OSEQ085 CagPromoter_fwd_SEQ
     primer_bind     3761..3775
                     /label=oKL260
     regulatory      3761..3770
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3767..4477
                     /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     3767..3784
                     /label=oCO03 mRuby2_fwd
     primer_bind     3767..3784
                     /label=oKL266
     primer_bind     complement(3793..3814)
                     /label=oEP076
     misc_feature    3813..3816
                     /label=splice site 1
     primer_bind     3815..3834
                     /label=oEP073
     primer_bind     3837..3856
                     /label=oSEQ062 mRuby2_fwd
     primer_bind     complement(3837..3856)
                     /label=oSEQ093 mRuby2_rev
     misc_feature    4035..4038
                     /label=splice site 2
     primer_bind     complement(4255..4274)
                     /label=oCO04 mRuby2_rev
     misc_feature    4313..4316
                     /label=splice site 3
     primer_bind     complement(4369..4388)
                     /label=oSEQ061 mRuby2_rev
     primer_bind     4369..4386
                     /label=oKL117
     primer_bind     4403..4422
                     /label=OSEQ091 mRuby2_fwd_SEQ
     primer_bind     complement(4457..4498)
                     /label=oKL303
                     /note="rev for PCR192"
     primer_bind     4478..4500
                     /label=oKL166
     primer_bind     4478..4499
                     /label=oKL141
     primer_bind     4478..4498
                     /label=oKL183
     primer_bind     complement(4478..4497)
                     /label=oKL182
     CDS             4487..4543
                     /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     4524..4563
                     /label=oKL250
     primer_bind     4524..4546
                     /label=oKL266
     primer_bind     complement(4526..4543)
                     /label=oKL202
     CDS             4544..5143
                     /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     4544..4565
                     /label=pPV2-puro_fwd
     primer_bind     complement(4544..4563)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4544..4563
                     /label=oSEQ047 phage-dest-downtream-seq
     primer_bind     complement(5009..5036)
                     /label=puro1_rev
     primer_bind     5017..5044
                     /label=puro2_fwd
     primer_bind     5040..5060
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     5109..5124
                     /label=OSEQ086 Puro_rev_SEQ
     primer_bind     complement(5123..5141)
                     /label=oKL300
                     /note="for PCR188 P2A-PuroR"
     primer_bind     complement(5123..5140)
                     /label=oKL066
     primer_bind     complement(5123..5140)
                     /label=oKL251
     primer_bind     5126..5167
                     /label=gebGH_fwd
     primer_bind     complement(5126..5147)
                     /label=pPV2-puro_rev
     primer_bind     5149..5170
                     /label=GG_bGH_fwd
     primer_bind     5153..5170
                     /label=GG_WPRE-bGH_fwd
     polyA_signal    5155..5379
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(join(5361..5379,1..4))
                     /label=gebGH_rev
     primer_bind     complement(5365..5379)
                     /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 cttactacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     2101 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     2161 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     2221 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     2281 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     2341 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     2401 gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc
     2461 tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg
     2521 gggggggggg gggggcgcgc gccrggsggg gsggggsggg gsgrggggsg gggsggggcg
     2581 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
     2641 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     2701 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
     2761 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
     2821 aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag
     2881 gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg
     2941 tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc
     3001 ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc
     3061 ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg
     3121 gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc
     3181 cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg
     3241 ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct
     3301 cgggccgggg rgggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga
     3361 ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt
     3421 cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc
     3481 gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt
     3541 gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac
     3601 ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc
     3661 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac
     3721 gtgctggtta ttgtgctgtc tcatcatttt ggcaaaaatg gccaccatgg tgtctaaggg
     3781 cgaagagctg atcaaggaaa atatgcgtat gaaggtggtc atggaaggtt cggtcaacgg
     3841 ccaccaattc aaatgcacag gtgaaggaga aggcaatccg tacatgggaa ctcaaaccat
     3901 gaggatcaaa gtcatcgagg gaggacccct gccatttgcc tttgacattc ttgccacgtc
     3961 gttcatgtat ggcagccgta cttttatcaa gtacccgaaa ggcattcctg atttctttaa
     4021 acagtccttt cctgagggtt ttacttggga aagagttacg agatacgaag atggtggagt
     4081 cgtcaccgtc atgcaggaca ccagccttga ggatggctgt ctcgtttacc acgtccaagt
     4141 cagaggggta aactttccct ccaatggtcc cgtgatgcag aagaagacca agggttggga
     4201 gcctaataca gagatgatgt atccagcaga tggtggtctg aggggataca ctcatatggc
     4261 actgaaagtt gatggtggtg gccatctgtc ttgctctttc gtaacaactt acaggtcaaa
     4321 aaagaccgtc gggaacatca agatgcccgg tatccatgcc gttgatcacc gcctggaaag
     4381 gttagaggaa agtgacaatg aaatgttcgt agtacaacgc gaacacgcag ttgccaagtt
     4441 cgccgggctt ggtggtggga tggacgagct gtacaaggga agcggagcta ctaacttcag
     4501 cctgctgaag caggctggag acgtggagga gaaccctgga cctatgaccg agtacaagcc
     4561 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     4621 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     4681 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     4741 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     4801 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     4861 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     4921 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     4981 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacat ccgcgccccg
     5041 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     5101 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacaacgtt gcacctgtgc
     5161 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     5221 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     5281 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     5341 acaatagcag gcatgctggg gatgcggtgg gctctatgg
//
