LOCUS       Exported                7113 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses human SOX2 and eGFP via t2A linker..
ACCESSION   .
VERSION     .
KEYWORDS    Sox2-t2A-GFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7113)
  AUTHORS   Dhaliwal NK, Abatti LE, Mitchell JA
  TITLE     KLF4 protein stability regulated by interaction with pluripotency 
            transcription factors overrides transcriptional control.
  JOURNAL   Genes Dev. 2019 Jun 20. pii: gad.324319.119. doi: 
            10.1101/gad.324319.119.
  PUBMED    31221664
REFERENCE   2  (bases 1 to 7113)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7113
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        74..453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        454..657
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     608..628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     654..678
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1713..1766
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1773..2486
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             1773..2486
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1815..1836)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2076..2095)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2423..2444
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2523..2542)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2567..2615
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2817..3245
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2904..2923)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3114..3135
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3254..3274)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3259..3588
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3439..3574
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3501..3520
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3655..4449
                     /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(3709..3728)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4319..4338
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4625..4746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4662..4681)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4716..4735
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4795..4811)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4795..4811)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4808..4830)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4819..4835
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(4843..4873)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4888..4909
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5026..5043)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5197..5785)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5277..5296)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5956..6816)
                     /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     6579..6598
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6817..6921)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6996..7015)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
       61 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      121 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      181 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      241 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      301 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      361 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      421 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      481 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      541 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      601 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      661 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      721 caccgggacc gatccagcct ccggactcta gaatgtacaa catgatggag acggagctga
      781 agccgccggg cccgcagcaa acttcggggg gcggcggcgg caactccacc gcggcggcgg
      841 ccggcggcaa ccagaaaaac agcccggacc gcgtcaagcg gcccatgaat gccttcatgg
      901 tgtggtcccg cgggcagcgg cgcaagatgg cccaggagaa ccccaagatg cacaactcgg
      961 agatcagcaa gcgcctgggc gccgagtgga aacttttgtc ggagacggag aagcggccgt
     1021 tcatcgacga ggctaagcgg ctgcgagcgc tgcacatgaa ggagcacccg gattataaat
     1081 accggccccg gcggaaaacc aagacgctca tgaagaagga taagtacacg ctgcccggcg
     1141 ggctgctggc ccccggcggc aatagcatgg cgagcggggt cggggtgggc gccggcctgg
     1201 gcgcgggcgt gaaccagcgc atggacagtt acgcgcacat gaacggctgg agcaacggca
     1261 gctacagcat gatgcaggac cagctgggct acccgcagca cccgggcctc aatgcgcacg
     1321 gcgcagcgca gatgcagccc atgcaccgct acgacgtgag cgccctgcag tacaactcca
     1381 tgaccagctc gcagacctac atgaacggct cgcccaccta cagcatgtcc tactcgcagc
     1441 agggcacccc tggcatggct cttggctcca tgggttcggt ggtcaagtcc gaggccagct
     1501 ccagcccccc tgtggttacc tcttcctccc actccagggc gccctgccag gccggggacc
     1561 tccgggacat gatcagcatg tatctccccg gcgccgaggt gccggaaccc gccgccccca
     1621 gcagacttca catgtcccag cactaccaga gcggcccggt gcccggcacg gccattaacg
     1681 gcacactgcc cctctcacac atgttggcta gcgagggcag aggaagtctt ctaacatgcg
     1741 gtgacgtgga ggagaatccc ggccctggta ccgtgagcaa gggcgaggag ctgttcaccg
     1801 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1861 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1921 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     1981 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     2041 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     2101 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     2161 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     2221 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     2281 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2341 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     2401 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2461 ctctcggcat ggacgagctg tacaagaccg gttagtaatg agtttaaacg ggggaggcta
     2521 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     2581 cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     2641 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     2701 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     2761 ggcggcaggc cctgccatag cagatctgcg cagctggggc tctagggggt atccccacgc
     2821 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2881 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2941 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3001 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3061 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3121 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3181 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3241 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3301 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3361 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3421 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3481 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     3541 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     3601 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     3661 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     3721 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     3781 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     3841 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     3901 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     3961 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4021 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     4081 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     4141 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     4201 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     4261 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4321 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4381 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4441 ttcttctgag cgggactctg gggttcgcga aatgaccgac caagcgacgc ccaacctgcc
     4501 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4561 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4621 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4681 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4741 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4801 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4861 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4921 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4981 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5041 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5101 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5161 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5221 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5281 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5341 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5401 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5461 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5521 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5581 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5641 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5701 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5761 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5821 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5881 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5941 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6001 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6061 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6121 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6181 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6241 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6301 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6361 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6421 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6481 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6541 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6601 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6661 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6721 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6781 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6841 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6901 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     6961 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     7021 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     7081 atttaagcta caacaaggca aggcttgacc gac
//
