LOCUS       sequence-393120-        7925 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses human codon-optimized Pseudoalteromonas TnsA-TnsB fusion 
            from a CMV promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7925)
  AUTHORS   Lampe GD, King RT, Halpin-Healy TS, Klompe SE, Hogan MI, Vo PLH, 
            Tang S, Chavez A, Sternberg SH
  TITLE     Targeted DNA integration in human cells without double-strand breaks
            using CRISPR-associated transposases.
  JOURNAL   Nat Biotechnol. 2023 Mar 29. doi: 10.1038/s41587-023-01748-1.
  PUBMED    36991112
REFERENCE   2  (bases 1 to 7925)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2023 Mar 29. doi: 10.1038/s41587-023-01748-1."
COMMENT     Sequence Label: pSL3402 (pTnsABf_Pse)
FEATURES             Location/Qualifiers
     source          1..7925
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..33
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(252..356)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(431..450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        622..1001
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1002..1205
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1156..1176
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1250..1269
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1250..1268
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1991..2011
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3903..3920)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3909..4133
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4179..4607
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4266..4285)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4476..4497
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4616..4636)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4621..4950
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4801..4936
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4863..4882
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5017..5811
                     /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(5071..5090)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5681..5700
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5985..6106
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6022..6041)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6076..6095
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6155..6171)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6155..6171)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6168..6190)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6179..6195
                     /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(join(6203..6209,6210..6227,6228..6233))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6248..6269
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6386..6403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6557..7145)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6637..6656)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7316..7925,1..182,183..251))
                     /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"
ORIGIN
        1 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
       61 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      121 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      181 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
      241 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
      301 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
      361 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
      421 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
      481 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
      541 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
      601 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
      661 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      721 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      781 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      841 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      901 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      961 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1021 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1081 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1141 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1201 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     1261 actataggga gacccaagct ggctagcgtt taaacttaag cttggtaccg ccaccatgta
     1321 catcagaaac ctgagaaagc cttctccaaa caagaacgtg ttcaagttcg cctccaccaa
     1381 ggtgtccagc gtggtgatgt gcgagagcag cctggagttc gacgcctgct tccaccacga
     1441 gtacaacgac cttatcgagt ctttcggcag ccaacctgag ggcttcaaat acgagttcat
     1501 gggcaagtcc ctgccttata cacctgatgc cctgatcagc tacaccgata agacacagaa
     1561 ataccacgag tacaagccct actctaagat cgccagccct ctgttccggg ctgaatttgc
     1621 cgccaagcgg gccgcttctc tgaaactggg aatcgacctg gtcctggtga ccgacagaca
     1681 gatcagagtg aaccctatcc tgaacaatct gaagctgctg catagataca gcggcgtgta
     1741 cggcatcagc ggcatccaga aggaactgct gagctttatc cacaaaagcg gcgttatcaa
     1801 gctgaatgac atcagcagcc aggtgggaat ccccatcggc gaaacccgga gcttcctgtt
     1861 tggcctcatg cacaagggcc tggtgaaggc cgacctgggc tgtgatgacc tgaccaacaa
     1921 ccccaccctg tgggccacac ctggcagcgg ctccggaaaa cggacagccg acggaagcga
     1981 gttcgagtca ccaaagaaga agcggaaagt cggatccggc tctggaggca tgaccgattt
     2041 ctttaacgag ttcgacgaga gcctggtgcc cctgaagcct cagaccccaa cacagtacgt
     2101 gaagctggac gatgccaacc tgatccagcg ggatctggac accttctccg ataccttcaa
     2161 gaatcaggcc ctgcagagat ataagctgat ctccaccatc gacaagaagc tgtctagggg
     2221 ctggacacag cgcaacctgg accccatcct ggacgagctg ttcaagggcg gcgacgtggt
     2281 gaggcctaat tggcgcacag tggcccggtg gagaaagaag tacatcgagt ctaacggcga
     2341 tatcgccagc ctggccgaca agaatcacaa gatgggcaac aggaccaatc gcatcaaggg
     2401 cgacgataag ttctttgata aggccctgga gcggttcctg gacgccaaga gacctacaat
     2461 cgccaccgcc tatcagtact ataaggatct gatcgtgatc gagaacgagt ccatcgtgga
     2521 gggcaagatc ccaatcatct cttacaacgc ctttaataag aggatcaagg ccatcccacc
     2581 ttatgcagtg gcagtggcac gccacggcaa gttcaaggcc gaccagtggt ttgcatactg
     2641 cgcagcacac gtgccaccaa ccagaatcct ggagcgggtg gagatcgatc acacacccct
     2701 ggacctgatc ctgctggacg atgagctgct gatcccaatc ggcaggccct atctgacact
     2761 gctgatcgac gtgttcagcg gctgcgtgct gggctttcac ctgagctaca agagcccttc
     2821 ctacgtgagc gccgcaaagg caatcaccca cgccatcaag ccaaagtccc tggatgccct
     2881 gaacatcgag ctgcagaatg actggccctg cttcggcaag tttgagaacc tggtggtgga
     2941 caatggcgcc gagttctggt ctaagaacct ggagcacgcc tgtcagagcg ccggcatcaa
     3001 catccagtac aatcctgtga ggaagccctg gctgaagccc tttatcgagc ggttcttcgg
     3061 cgtgatgaac gagtacttcc tgcccgagct gcctggcaag accttttcta atatcctgga
     3121 gaaggaggag tataagcccg agaaggacgc catcatgcgg ttcagcacat ttgtggagga
     3181 gttccacaga tggatcgccg acgtgtacca ccaggactct aatagccggg agaccagaat
     3241 ccctatcaag aggtggcagc agggcttcga tgcctatcct ccactgacca tgaacgagga
     3301 ggaggagaca aggttttcca tgctgatgcg catctccgac tctaggaccc tgacacgcaa
     3361 tggctttaag taccaggagc tgatgtatga ttctaccgcc ctggccgact accgcaagca
     3421 ctatccacag acaaaggaga ccgtgaagaa gctgatcaag gtggaccccg acgatatcag
     3481 caaaatctac gtgtacctgg aggagctgga gtcctacctg gaggtgcctt gcacagatcc
     3541 aaccggctac acagacggcc tgagcatcta tgagcacaag accatcaaga agatcaaccg
     3601 ggaagtgatc agagagagca aggactccct gggcctggcc aaggcaagga tggccatcca
     3661 cgagagagtg aagcaggagc aggaggtgtt catcgagtcc aagaccaagg ccaagatcac
     3721 agccgtgaag aagcaggccc agatcgccga tgtgagcaat accggcacat ccaccatcaa
     3781 ggtgtctgag gagagcgccg caccagtgca gaagcacatc tccaacgaca attctgacga
     3841 ttgggacgat gacctggagg cctttgagtg atctagaggg cccgtttaaa cccgctgatc
     3901 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3961 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     4021 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     4081 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     4141 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     4201 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4261 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4321 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4381 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4441 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4501 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     4561 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     4621 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4681 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     4741 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4801 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4861 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     4921 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     4981 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     5041 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     5101 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     5161 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     5221 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     5281 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     5341 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     5401 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     5461 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     5521 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     5581 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     5641 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     5701 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     5761 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     5821 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     5881 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     5941 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     6001 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     6061 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     6121 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     6181 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     6241 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     6301 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6361 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     6421 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     6481 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6541 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6601 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6661 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6721 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6781 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6841 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6901 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6961 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     7021 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     7081 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     7141 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     7201 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     7261 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     7321 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     7381 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     7441 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     7501 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     7561 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7621 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7681 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7741 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7801 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7861 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7921 gcccg
//
