LOCUS       Exported                7673 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Human transferrin receptor tagged with pH sensitive red fluorescent 
            protein pHuji.
ACCESSION   .
VERSION     .
KEYWORDS    TfR-pHuji
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7673)
  AUTHORS   Shen Y, Rosendale M, Campbell RE, Perrais D
  TITLE     pHuji, a pH-sensitive red fluorescent protein for imaging of exo- 
            and endocytosis.
  JOURNAL   J Cell Biol. 2014 Nov 10;207(3):419-32. doi: 10.1083/jcb.201404107.
  PUBMED    25385186
REFERENCE   2  (bases 1 to 7673)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7673
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     815..832
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             complement(1862..1873)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3218..3925
                     /codon_start=1
                     /product="pH-sensitive red fluorescent protein derived from
                     mApple, for imaging of exocytosis and endocytosis (Shen et 
                     al., 2014)"
                     /label=pHuji
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEA
                     FQTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNF
                     EDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALK
                     SEIKYRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     3631..3650
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     regulatory      3960..3969
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     polyA_signal    3973..4107
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4010..4029)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4064..4083
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4176..4533
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(4199..4219)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4384..4519
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4446..4465
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(4553..4570)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4553..4569)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4562..4584)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4782..5237
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4869..4888)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5079..5100
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5254..5273)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5373..5395
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5433..5451)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5519..5623
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5624..6484
                     /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(5842..5861)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6655..7243
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7144..7163
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7397..7414
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7531..7552
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7567..7597
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7605..7621
                     /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)."
     primer_bind     7610..7632
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7629..7645
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7629..7645
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
      901 cgattgaatt ccccggggat ccaccatgga tcaagctaga tcagcattct ctaacttgtt
      961 tggtggagaa ccattgtcat atacccggtt cagcctggct cggcaagtag atggcgataa
     1021 cagtcatgtg gagatgaaac ttgctgtaga tgaagaagaa aatgctgaca ataacacaaa
     1081 ggccaatgtc acaaaaccaa aaaggtgtag tggaagtatc tgctatggga ctattgctgt
     1141 gatcgtcttt ttcttgattg gatttatgat tggctacttg ggctattgta aaggggtaga
     1201 accaaaaact gagtgtgaga gactggcagg aaccgagtct ccagtgaggg aggagccagg
     1261 agaggacttc cctgcagcac gtcgcttata ttgggatgac ctgaagagaa agttgtcgga
     1321 gaaactggac agcacagact tcaccagcac catcaagctg ctgaatgaaa attcatatgt
     1381 ccctcgtgag gctggatctc aaaaagatga aaatcttgcg ttgtatgttg aaaatcaatt
     1441 tcgtgaattt aaactcagca aagtctggcg tgatcaacat tttgttaaga ttcaggtcaa
     1501 agacagcgct caaaactcgg tgatcatagt tgataagaac ggtagacttg tttacctggt
     1561 ggagaatcct gggggttatg tggcgtatag taaggctgca acagttactg gtaaactggt
     1621 ccatgctaat tttggtacta aaaaagattt tgaggattta tacactcctg tgaatggatc
     1681 tatagtgatt gtcagagcag ggaaaatcac ctttgcagaa aaggttgcaa atgctgaaag
     1741 cttaaatgca attggtgtgt tgatatacat ggaccagact aaatttccca ttgttaacgc
     1801 agaactttca ttctttggac atgctcatct ggggacaggt gacccttaca cacctggatt
     1861 cccttccttc aatcacactc agtttccacc atctcggtca tcaggattgc ctaatatacc
     1921 tgtccagaca atctccagag ctgctgcaga aaagctgttt gggaatatgg aaggagactg
     1981 tccctctgac tggaaaacag actctacatg taggatggta acctcagaaa gcaagaatgt
     2041 gaagctcact gtgagcaatg tgctgaaaga gataaaaatt cttaacatct ttggagttat
     2101 taaaggcttt gtagaaccag atcactatgt tgtagttggg gcccagagag atgcatgggg
     2161 ccctggagct gcaaaatccg gtgtaggcac agctctccta ttgaaacttg cccagatgtt
     2221 ctcagatatg gtcttaaaag atgggtttca gcccagcaga agcattatct ttgccagttg
     2281 gagtgctgga gactttggat cggttggtgc cactgaatgg ctagagggat acctttcgtc
     2341 cctgcattta aaggctttca cttatattaa tctggataaa gcggttcttg gtaccagcaa
     2401 cttcaaggtt tctgccagcc cactgttgta tacgcttatt gagaaaacaa tgcaaaatgt
     2461 gaagcatccg gttactgggc aatttctata tcaggacagc aactgggcca gcaaagttga
     2521 gaaactcact ttagacaatg ctgctttccc tttccttgca tattctggaa tcccagcagt
     2581 ttctttctgt ttttgcgagg acacagatta tccttatttg ggtaccacca tggacaccta
     2641 taaggaactg attgagagga ttcctgagtt gaacaaagtg gcacgagcag ctgcagaggt
     2701 cgctggtcag ttcgtgatta aactaaccca tgatgttgaa ttgaacctgg actatgagag
     2761 gtacaacagc caactgcttt catttgtgag ggatctgaac caatacagag cagacataaa
     2821 ggaaatgggc ctgagtttac agtggctgta ttctgctcgt ggagacttct tccgtgctac
     2881 ttccagacta acaacagatt tcgggaatgc tgagaaaaca gacagatttg tcatgaagaa
     2941 actcaatgat cgtgtcatga gagtggagta tcacttcctc tctccctacg tatctccaaa
     3001 agagtctcct ttccgacatg tcttctgggg ctccggctct cacacgctgc cagctttact
     3061 ggagaacttg aaactgcgta aacaaaataa cggtgctttt aatgaaacgc tgttcagaaa
     3121 ccagttggct ctagctactt ggactattca gggagctgca aatgccctct ctggtgacgt
     3181 ttgggacatt gacaatgagt ttggggatcc accggtcatg gtgagcaagg gcgaggagaa
     3241 taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa
     3301 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg cctttcagac
     3361 cgctaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc
     3421 tcagttcatg tacggctcca aggtctacat taagcaccca gccgacatcc ccgactactt
     3481 caagctgtcc ttccccgagg gcttcaggtg ggagcgcgtg atgaacttcg aggacggcgg
     3541 cattattcac gttaaccagg actcctccct gcaggacggc gtgttcatct acaaggtgaa
     3601 gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg
     3661 ggaggcctcc gaggagcgga tgtaccccga ggacggcgcc ctgaagagcg agattaagta
     3721 taggctgaag ctgaaggacg gcggccacta cgccgccgag gtcaagacca cctacaaggc
     3781 caagaagccc gtgcagctgc ccggcgccta catcgtggac atcaagttgg acatcgtgtc
     3841 ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac
     3901 cggcggcatg gacgagctgt acaaggctag ctaatctaga gtcgacctgc agaagcttgg
     3961 ccgccatggc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4021 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4081 atcaatgtat cttatcatgt ctggatcggg aattaattcg gcgcagcacc atggcctgaa
     4141 ataacctctg aaagaggaac ttggttaggt accttctgag gcggaaagaa ccagctgtgg
     4201 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4261 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     4321 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     4381 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4441 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     4501 ggaggctttt ttggaggcct aggcttttgc aaaaagctgt taacagcttg gcactggccg
     4561 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     4621 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     4681 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     4741 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4801 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4861 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4921 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4981 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5041 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5101 aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc
     5161 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5221 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5281 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5341 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5401 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5461 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5521 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5581 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5641 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5701 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5761 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5821 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5881 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5941 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6001 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6061 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6121 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6181 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6241 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6301 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6361 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6421 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6481 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6541 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6601 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6661 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6721 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6781 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6841 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6901 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6961 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7021 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7081 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7141 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7201 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7261 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7321 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7381 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     7441 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     7501 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     7561 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     7621 atttcacaca ggaaacagct atgacatgat tacgaattaa ttcgagctcg ccc
//
