LOCUS Exported 8244 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION To convert human fetal lung fibroblasts into induced cholinergic neurons (hiCN) in combination with two small molecules, forskolin and dorsomorphin.. ACCESSION . VERSION . KEYWORDS pLNCX2-NGN2-IRES-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8244) AUTHORS Liu ML, Zang T, Zou Y, Chang JC, Gibson JR, Huber KM, Zhang CL TITLE Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons. JOURNAL Nat Commun. 2013;4:2183. doi: 10.1038/ncomms3183. PUBMED 23873306 REFERENCE 2 (bases 1 to 8244) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8244 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 23..326 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 327..530 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 481..501 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" misc_feature 1431..1982 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(1575..1592) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 1802..1821 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 1985..2704 /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 1985..2701 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2030..2051) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2291..2310) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2638..2659 /label=EGFP-C /note="EGFP, forward primer" LTR 2792..3385 /note="long terminal repeat from Moloney murine leukemia virus" primer_bind complement(3488..3510) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 3596..3736 /label=bom /note="basis of mobility region from pBR322" primer_bind 3651..3670 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(3751..3768) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3922..4510) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4002..4021) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4681..5541) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 5304..5323 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5542..5646) /gene="bla" /label=AmpR promoter primer_bind 5714..5732 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" LTR 6304..6479 /label=5' LTR (truncated) /note="truncated long terminal repeat from Moloney murine sarcoma virus" misc_feature 6542..6741 /label=MMLV Psi /note="packaging signal of Moloney murine leukemia virus (MMLV)" CDS 6942..7358 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind 7288..7310 /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" primer_bind 7326..7342 /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" CDS 7402..8196 /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(7456..7475) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 8066..8085 /label=Neo-F /note="Neomycin resistance gene, forward primer" ORIGIN 1 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 61 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 121 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 181 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 241 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 301 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 361 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 421 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 481 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga 541 accgtcagat ccgctagcgc taccggactc agatccaccg gtgccaccat gttcgtcaaa 601 tccgagacct tggagttgaa ggaggaagag gacgtgttag tgctgctcgg atcggcctcc 661 cccgccttgg cggccctgac cccgctgtca tccagcgccg acgaagaaga ggaggaggag 721 ccgggcgcgt caggcggggc gcgtcggcag cgcggggctg aggccgggca gggggcgcgg 781 ggcggcgtgg ctgcgggtgc ggagggctgc cggcccgcac ggctgctggg tctggtacac 841 gattgcaaac ggcgcccttc ccgggcgcgg gccgtctccc gaggcgccaa gacggccgag 901 acggtgcagc gcatcaagaa gacccgtaga ctgaaggcca acaaccgcga gcgaaaccgc 961 atgcacaacc tcaacgcggc actggacgcg ctgcgcgagg tgctccccac gttccccgag 1021 gacgccaagc tcaccaagat cgagaccctg cgcttcgccc acaactacat ctgggcactc 1081 accgagaccc tgcgcctggc ggatcactgc gggggcggcg gcgggggcct gccgggggcg 1141 ctcttctccg aggcagtgtt gctgagcccg ggaggcgcca gcgccgccct gagcagcagc 1201 ggagacagcc cctcgcccgc ctccacgtgg agttgcacca acagccccgc gccgtcctcc 1261 tccgtgtcct ccaattccac ctccccctac agctgcactt tatcgcccgc cagcccggcc 1321 gggtcagaca tggactattg gcagccccca cctcccgaca agcaccgcta tgcacctcac 1381 ctccccatag ccagggattg tatctagatg aattccgccc ccccccccta acgttactgg 1441 ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt 1501 gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc 1561 taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc 1621 agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg 1681 gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc 1741 tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa 1801 atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg 1861 tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa 1921 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa 1981 taccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 2041 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 2101 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 2161 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat 2221 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 2281 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac 2341 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 2401 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa 2461 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 2521 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 2581 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 2641 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa 2701 gtaaagcggc cgcgactcta gagtcgagtt tccttaatgg cctaacatcg ataaaataaa 2761 agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca 2821 agctagctta agtaacgcca ttttgcaagg catggaaaaa tacataactg agaatagaga 2881 agttcagatc aaggtcagga acagatggaa cagctgaata tgggccaaac aggatatctg 2941 tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggaacagct gaatatgggc 3001 caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc 3061 cccagatgcg gtccagccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc 3121 ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct 3181 tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggg 3241 gcgccagtcc tccgattgac tgagtcgccc gggtacccgt gtatccaata aaccctcttg 3301 cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac 3361 tacccgtcag cgggggtctt tcatttgggg gctcgtccgg gatcgggaga cccctgccca 3421 gggaccaccg acccaccacc gggaggtaag ctggctgcct cgcgcgtttc ggtgatgacg 3481 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg 3541 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag 3601 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga 3661 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag 3721 aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 3781 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 3841 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 3901 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 3961 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 4021 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 4081 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 4141 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 4201 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 4261 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 4321 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg 4381 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 4441 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 4501 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 4561 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 4621 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 4681 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 4741 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 4801 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 4861 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 4921 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 4981 cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 5041 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 5101 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 5161 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 5221 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 5281 ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct 5341 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 5401 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 5461 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 5521 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 5581 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 5641 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac 5701 attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaat tcataccaga 5761 tcaccgaaaa ctgtcctcca aatgtgtccc cctcacactc ccaaattcgc gggcttctgc 5821 ctcttagacc actctaccct attccccaca ctcaccggag ccaaagccgc ggcccttccg 5881 tttctttgct tttgaaagac cccacccgta ggtggcaagc tagcttaagt aacgccactt 5941 tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag gtcaggaaca 6001 aagaaacagc tgaataccaa acaggatatc tgtggtaagc ggttcctgcc ccggctcagg 6061 gccaagaaca gatgagacag ctgagtgatg ggccaaacag gatatctgtg gtaagcagtt 6121 cctgccccgg ctcggggcca agaacagatg gtccccagat gcggtccagc cctcagcagt 6181 ttctagtgaa tcatcagatg tttccagggt gccccaagga cctgaaaatg accctgtacc 6241 ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc cgctctccga 6301 gctcaataaa agagcccaca acccctcact cggcgcgcca gtcttccgat agactgcgtc 6361 gcccgggtac ccgtattccc aataaagcct cttgctgttt gcatccgaat cgtggtctcg 6421 ctgttccttg ggagggtctc ctctgagtga ttgactaccc acgacggggg tctttcattt 6481 gggggctcgt ccgggatttg gagacccctg cccagggacc accgacccac caccgggagg 6541 taagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg tttgatgtta 6601 tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa 6661 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc 6721 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg 6781 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt 6841 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt 6901 gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc 6961 ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg 7021 tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg 7081 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg 7141 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag 7201 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc 7261 ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat 7321 cctcccttta tccagccctc actccttctc taggcgccgg aattccgatc tgatcaagag 7381 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc 7441 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat 7501 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg 7561 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg 7621 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta 7681 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta 7741 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc 7801 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc 7861 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg 7921 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg 7981 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt 8041 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc 8101 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc 8161 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaatagt 8221 aatcaattac ggggtcatta gttc //