LOCUS       sequence-80504-e        6769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  a bicistronic IRES-mAmetrine 1.1 containing vector for mammalian 
            expression (CMV promoter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6769)
  TITLE     Vignali lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6769)
  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            "Unpublished"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: pIAneo
FEATURES             Location/Qualifiers
     source          1..6769
                     /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    1136..1700
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1151..1702
                     /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(1705..1707,1708..1710,1711..2424)
                     /codon_start=1
                     /product="photostable yellow fluorescent protein with a 
                     large Stokes shift, excitable with violet light (Ai et al.,
                     2008)"
                     /label=mAmetrine1.1
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVRGEGEGDATNGKLTL
                     KFICTSGELPVPWPTLVTTLSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GSYRTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNMNAWDAYITADKQKNGIK
                     ANFKIEHNVKDGGVQLADAYQQNTPIGDGSVLLPDNHYLSFQSKLFKDPNEQRDHMVLL
                     EFVTAAGITPGMDELYK"
     primer_bind     complement(1750..1771)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    2472..2593
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2483..2502)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2537..2556
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2779..3234
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2866..2885)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3076..3097
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3351..3708
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3351..3542
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3374..3394)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3559..3694
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3759..4553
                     /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(3813..3832)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4423..4442
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4617..4665
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4707..4726)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4826..4848
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4886..4904)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4972..5076
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5077..5145,5146..5937)
                     /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(5295..5314)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6108..6696
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6597..6616
                     /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 aaacgtctag gccccccgaa ccacggggac gtggttttcc
     1681 tttgaaaaac acgatgataa taccatggtg agcaagggcg aggagctgtt caccggggtg
     1741 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaggttcag cgtgcgcggc
     1801 gagggcgagg gcgatgccac caacggcaag ctgaccctga agttcatctg cacctccggc
     1861 gagctgcccg tgccctggcc caccctcgtg accaccctgt cttacggcgt gcagtgcttc
     1921 gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1981 tacgtccagg agcgcaccat ctccttcaag gacgacggca gctacaggac ccgcgccgag
     2041 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     2101 gaggacggca acatcctggg gcacaagctg gagtacaaca tgaacgcgtg ggacgcgtat
     2161 atcacggccg acaagcagaa gaacggcatc aaagcgaact tcaagatcga gcacaacgtc
     2221 aaggacggcg gcgtgcagct cgccgacgcg taccagcaga acacccccat cggcgacggc
     2281 tccgtgctgc tgcctgacaa ccactacctg agcttccaga gcaagctgtt caaagacccc
     2341 aacgagcagc gcgatcacat ggtcctgctg gagttcgtta ccgccgccgg gataactccc
     2401 ggcatggacg agctgtacaa gtaaagcggc cgcttccctt tagtgagggt taatgcttcg
     2461 agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     2521 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     2581 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggagat
     2641 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcc gataaggatc
     2701 gatccgggct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     2761 agcctgaatg gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2821 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2881 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2941 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3001 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3061 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3121 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3181 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat
     3241 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgcg gatctgcgca
     3301 gcaccatggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga
     3361 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3421 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3481 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3541 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3601 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     3661 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gcttgattct
     3721 tctgacacaa cagtctcgaa cttaaggcta gagccaccat gattgaacaa gatggattgc
     3781 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3841 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3901 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     3961 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     4021 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     4081 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     4141 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     4201 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4261 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     4321 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4381 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4441 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4501 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4561 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgat ggccgcaata
     4621 aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgaatcg atagcgataa
     4681 ggatcgcgcg tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4741 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4801 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4861 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     4921 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4981 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5041 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5101 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5161 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5221 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5281 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5341 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5401 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5461 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5521 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5581 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5641 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5701 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5761 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     5821 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5881 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5941 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6001 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6061 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6121 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6181 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6241 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6301 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6361 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6421 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6481 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6541 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6601 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6661 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6721 cggttcctgg ccttttgctg gccttttgct cacatggctc gacagatct
//
