LOCUS       sequence-206319-        6766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  a bicistronic IRES-GFP containing vector for mammalian expression 
            (CMV promoter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6766)
  TITLE     Vignali lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6766)
  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            "Unpublished"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: pIGneo
FEATURES             Location/Qualifiers
     source          1..6766
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     680..700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1005..1024
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1067..1085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    1151..1700
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1295..1312)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1522..1541
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1703..1705,1706..1708,1709..2422)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1703..1705,1706..1708,1709..2419)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1748..1769)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2009..2028)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2356..2377
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2470..2591
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2481..2500)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2535..2554
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2777..3232
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2864..2883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3074..3095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3349..3706
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3349..3540
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3372..3392)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3557..3692
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3757..4551
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3811..3830)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4421..4440
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4615..4663
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4704..4723)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4823..4845
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4883..4901)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4969..5073
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5074..5142,5143..5934)
                     /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(5292..5311)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6105..6693
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6594..6613
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
      661 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact
     1081 ataggctagc ctcgagaatt cacgcgtggt acctctagag tcgagcggga tcaattccgc
     1141 ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat
     1201 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct
     1261 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg
     1321 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta
     1381 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag
     1441 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg
     1501 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat
     1561 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca
     1621 tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc
     1681 ctttgaaaaa cacgataata ccatggtgag caagggcgag gagctgttca ccggggtggt
     1741 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1801 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1861 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     1921 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1981 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     2041 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     2101 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     2161 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     2221 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     2281 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     2341 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     2401 catggacgag ctgtacaagt aaagcggccg cttcccttta gtgagggtta atgcttcgag
     2461 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     2521 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     2581 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt
     2641 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatccga taaggatcga
     2701 tccgggctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     2761 cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2821 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2881 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2941 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3001 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3061 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     3121 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     3181 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttcctgatgc
     3241 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgcgga tctgcgcagc
     3301 accatggcct gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa
     3361 gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3421 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     3481 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     3541 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     3601 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     3661 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttgattcttc
     3721 tgacacaaca gtctcgaact taaggctaga gccaccatga ttgaacaaga tggattgcac
     3781 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3841 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3901 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     3961 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4021 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4081 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4141 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4201 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4261 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4321 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4381 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4441 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4501 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4561 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgatgg ccgcaataaa
     4621 atatctttat tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agcgataagg
     4681 atccgcgtat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     4741 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4801 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4861 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     4921 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4981 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5041 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5101 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5161 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5221 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5281 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5341 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5401 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5461 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5521 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5581 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5641 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5701 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5761 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     5821 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     5881 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5941 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6001 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6061 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6121 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6181 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6241 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6301 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6361 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6421 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6481 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6541 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6601 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6661 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6721 ttcctggcct tttgctggcc ttttgctcac atggctcgac agatct
//
